Gordon C. Balch

Alterations to gonadal development and reproductive success in japanese medaka (Oryzias latipes) exposed to 17α‐ethinylestradiol

The Japanese medaka (Oryzias latipes) was used as an in vivo model to evaluate the effects of exposure to the synthetic estrogen 17alpha-ethinylestradiol (EE2) on reproductive behavior and reproductive success in fish. Exposures to EE2 began within 2 to 5 d posthatch and continued until medaka were sexually mature, between four and six months of age. Among male and female medaka exposed to EE2 at nominal concentrations of 0.2 and 2 ng/L, mating behavior and reproductive success were normal in reproductive trials. However, reproductive behavior (i.e., copulations) was suppressed in the treatment with 10 ng/L EE2. Among 19 males exposed to 10 ng/L EE2 and placed with unexposed females in reproductive trials, 16 males did not copulate and reproductive success was very low. None of the females exposed to 10 ng/L of EE2 participated in reproductive behavior with unexposed males. The reproductive trials in combination with a histological survey indicated that male fish with gonadal intersex (i.e., testis-ova) were still capable of reproductive behavior and could fertilize eggs. Even though females exposed to 10 ng/L EE2 had poor reproductive success, their ovaries showed normal development and oogenesis. These data are relevant to observations of intersex in feral fish populations. Although intersex in male fish may be an indicator of exposure to estrogenic compounds, it appears that the presence of oocytes in testicular tissue may not directly impact the reproductive capability of the male fish. However, it is clear that concentrations of endocrine disrupting chemicals (EDCs) that cause gonadal intersex are sufficient to reduce reproductive performance.

Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes)

This study was focused on determining the effects of exposure to antiandrogens on the gonadal development of Japanese medaka (Oryzias latipes). Test compounds included the fungicide, vinclozolin and the clinical antiandrogen, cyproterone acetate. Newly hatched medaka were exposed to aqueous solutions of vinclozolin (2500 microg/l) and the vinclozolin fungicide formulation, Ronilan (1000 and 5000 microg/l) and cyproterone acetate (1 and 10 microg/l), for 3 months. Histological evaluation of the gonadal tissues of exposed fish indicated that the 5000 microg/l concentration of the vinclozolin formulation (Ronilan) induced a low incidence of intersex (i.e. testis-ova) and the 2500 microg/l concentration of vinclozolin-affected spermatogenesis in males. Also, the vinclozolin treatments induced moderate ovarian atresia. Cyproterone acetate also induced a low incidence of testis-ova, but in contrast to the vinclozolin treatment the amount of ovarian tissue in the testis-ova was equal to or greater than the amount of testicular tissue. In the cyproterone acetate treatments, both oogenesis and spermatogenesis were moderately inhibited at all test concentrations. The results of this study indicate that antiandrogens have the potential to alter testicular development and gametogenesis in fish. However, research is needed to determine the mechanisms by which antiandrogens affect fish.

Increased kidney, liver, and testicular cell death after chronic exposure to 17α-ethinylestradiol in medaka (Oryzias latipes)

Sublethal effects observed in fish exposed to environmental estrogens may be mediated via stimulation of cell death. To investigate whether cell death is induced in fish after chronic exposure to estrogenic chemicals, Japanese medaka (Oryzias latipes) were exposed from hatch until sexual maturity to 10 ng/L 17alpha-ethinylestradiol (EE2) or acetone solvent (control). Cell death was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl-mediated dUTP nick end-labeling (TUNEL), which labels nuclei of cells containing apoptotic or necrotic (fragmented) DNA. The major impact of EE2 exposure in both male and female medaka was to significantly increase the number of TUNEL-positive hepatocytes and kidney tubule cells compared to control. Cell morphology was consistent with apoptosis in the liver and cloudy swelling or necrosis in the tubule cells. The number of TUNEL-positive interstitial (hematopoietic) and glomerular cells was significantly greater in the kidneys of EE2-exposed male, but not female, medaka. The EE2 exposure also significantly increased the number of TUNEL-positive testicular cells in medaka compared to corresponding controls, namely Leydig cells, Sertoli cells, spermatocytes, and spermatids. In medaka with gonadal intersex, areas of fibrosis and areas containing female gonadal cells were relatively unstained with TUNEL. No effect of EE2 exposure on the number of TUNEL-positive ovarian somatic cells or on the rate of female ovarian follicle atresia was found. These results suggest that chronic exposure to EE2 in medaka is hepatotoxic and nephrotoxic in both sexes, whereas gonadal toxicity is specific to males.

Feminization of female leukophore‐free strain of Japanese medaka (Oryzias latipes) exposed to 17β‐estradiol

The recently developed female leukophore-free (FLFII) strain of Japanese medaka (Oryzias latipes) carries DNA markers for the identification of genotypic sex. Information regarding genotypic sex is useful for tests in which endocrine-disrupting compounds may masculinize or feminize fish. In the present study, methods were developed to automate DNA extraction and profiling for rapid determination of genotypic sex. Adequate amounts of DNA were isolated by robotic extraction procedures from the caudal fin. New primers were developed to include an 18-base pair segment that is in the X chromosome of female medaka but is absent in the Y chromosome of male medaka. Automated profiling methods with 96-well plates permitted analysis of the genomic sex of medaka at rates of up to 500 fish/d. We investigated the sensitivity of the FLFII strain to the feminizing effects of the potent estrogen 17beta-estradiol (E2), and we compared this sensitivity to that of a wild strain that has been used widely in the study of endocrine-disrupting compounds. All FLFII medaka exposed to 1 microg/L of E2 (n = 50) had the female gonadal phenotype (i.e., ovaries), and all but one wild-strain medaka exposed to 1 microg/L of E2 (i.e., 47 of 48 fish) had the female gonadal phenotype, indicating that the FLFII and wild strains have approximately equal sensitivities to the feminizing effects of E2. Analysis of the genotype of FLFII medaka confirmed that 100% of fish with the male genotype had been feminized to the female gonadal phenotype. The FLFII strain is an excellent teleost model for detecting feminization or masculinization of fish, and automated methods can be used for rapid analysis of the genotypic sex of FLFII medaka.

Zinc in plants, sediments, snow and ice around a galvanized electrical transmission tower in a beaver pond

Elevated concentrations of Zn in beaver pond sediments and in shoots and roots of Sparganium androcladum growing near a 25-yr old galvanized steel tower supporting a 230-kV electrical transmission line illustrated the point source nature of such towers to the aquatic environment. Concentrations of Zn in plants and sediments in the pond decreased exponentially with distance downstream from the tower and declined to background levels within a distance of 15 to 30 m from the tower. Snow and ice beneath this tower and another one, located in a terrestrial environment, also were contaminated with Zn. The distribution of Cd and Cu in the beaver pond sediments did not indicate a point source for these metals. Towers of the type investigated in this study can be coated with 23 to 133 kg of Zn, depending on their configuration. Substantially larger towers are used to support 500-kV transmission lines, suggesting that effects of Zn lost by corrosion from galvanized towers on biota in poorly buffered aquatic habitats may need further investigation. Then, if necessary, recommendations can be developed concerning the placement of galvanized steel towers near to or in susceptible aquatic habitats.