Title Studies on the evaluation of the effect of endocrine disrupting chemicals using transgenic see-through medaka (Oryzias latipes), olvas-GFP/STII-YI strain
Authers Takeshi HANO
Keywords transgenic medaka, endocrine disrupting chemicals, germ cell, Green fluorescent protein, sexual differentiation
Citation Bull. Fish. Res. Agen. No.36, 1-56, 2012
In the past few years, general concerns about the release of endocrine disrupting chemicals (EDCs) into the environment have increased because of their potential to cause adverse physiological effects in wildlife. In particular, estrogenic potential of certain chemicals have been shown to interfere with the sexual development and reproduction of fish. Evaluation of chemicals using intact fish is an important and available approach for monitoring and assessing the risks of chemical exposure, however, it requires labor-intensive, expensive, and time-consuming work. To solve this problem, an approach using transgenic fish has been proposed as a facilitated screening test model to evaluate the effect of EDCs because it may have the potential to provide in vivo biomonitoring and on-time and on-site evaluation of the effects of EDCs. This study was performed to evaluate the effects of estrogenic chemicals using transgenic see-through medaka (Oryzias latipes), olvas-GFP/STII-YI strain, which contains the green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene, and germ cell-specific expression of GFP can be visualized in living individuals. We exposed transgenic medaka to known doses of estrogen-mimicking chemicals and examined whether the effects on sexual differentiation including germ cell proliferation and matured gonad could be detected using GFP fluorescence.
 Prior to the chemical exposure, we had observed that the number of GFP-fluorescent germ cells and area of GFP fluorescence in normal XX females was about 10 times that in normal XY males at 10 days posthatch (dph). These results showed that sexual dimorphism could be detected on approximately the day of hatching using GFP fluorescence, and further indicated that alterations in sexual differentiation by EDCs could be determined by just with a single slice of image of gonads as early as 10days.
 In the first chemical exposure, we examined the effect of 17α-ethinyletradiol (EE2), a representative estrogen-mimicking chemical, on sexual differentiation after in ovo and waterborne exposures. In ovo exposure was performed using a nanoinjection method in which chemicals were directly injected into the embryo. Embryos (within 8 h after fertilization) were nanoinjected with 0.1, 0.5, 2.5, or 5.0 ng of EE2 and results indicated that some 10-dph, XY males from embryos injected with 0.5 ng EE2 showed a larger fluorescent area and more germ cells than those of control. At 100 dph, complete male to female sex reversal occurred in ≧ 0.5 ng treatments. In waterborne exposure experiment, from 0 dph onwards, juveniles were exposed to graded concentrations of EE2 (25.2, 45.1, 80.1, 158, 447, 880, or 1710 ng/L) for 35 days. The gonadal size of 10 dph males that had been exposed to 158 ng/L of EE2 significantly increased up to twice the size of control males. At 35dph, male to female sex reversal occurred at EE2 exposure ≧ 45.1 ng/L. These results suggest that enhanced germ cell proliferation in males indicated the occurrence of abnormal sexual differentiation toward females and that observation of proliferative activity of germ cells by GFP fluorescence can be applied to facilitated screening fish model to detect adverse effects on sexual differentiation as early as 10 dph juveniles.
 To assess further potential of the olvas-GFP/STII-YI strain,mature medaka at 60 dph were exposed to EE2 (47.8, 94.8, 216, or 522 ng/L) for 4 weeks. The gonads showed a significant reduction of the GFP-fluorescent area in males exposed to EE2 at > 216 ng/L and histologically, high connective tissue prevalence were observed at ≧ 216 ng/L. Next, mature male medaka were exposed to EE2 (43.7 85.8, 215, or 473 ng/L) for 3 weeks and allowed to depurate for 6 weeks, to investigate persistent effects of EE2. Continuous gonad observation showed that GFP began to decline 3 weeks after initial exposure to ≧ 215 ng/L. After depuration, the gonad’s fluorescent areas gradually recovered, with no statistical difference at the end of the depuration period; normal spermatogenesis was present in these individuals. These results showed that the condition of the gonad can clearly be described by GFP fluorescence and that abnormal changes of the gonad can be detected by just with a single slice of images of the gonad even in 60dph adult medaka.
 Overall, the present study clearly showed that the transgenic line test model can provide a more practical choice of the evaluation for the effect of EDCs and could be a promising noninvasive approach to identify the effects of chemicals on the gonad. Although we investigated a few chemicals only, further studies to investigate a variety of chemicals will elucidate the usefulness of olvas-GFP/STII-YI strain as a facilitated screening fish model to detect EDC-induced abnormalities.
URI http://www.fra.affrc.go.jp/bulletin/bull/bull36/36-1.pdf