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(292) Rapid assay for detecting the alteration of sexual differentiation caused by ethinylestradiol using transgenic medaka.
Hano, Takeshi*,1, Oshima, Yuji1, Oe, Toshiaki1, Kinoshita, Masato2, Tanaka, Minoru3, Wakamatsu, Yuko4, Ozato, Kenjiro4, Honjo, Tsuneo1, 1 Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka, Japan2 Graduate School of Agriculture, Kyoto Univesity, Kyoto, Japan3 Graduate School of Science, Hokkaido University, Sapporo, Japan4 Labolatory of Freshcenter Fish Stocks, Bioscience center, Nagoya Univesity, Nagoya, Japan
ABSTRACT- Endocrine-disrupting chemicals can interfere with the sexual differentiation of organisms. We investigated the alteration of sexual differentiation by such chemicals using olvas-GFP /ST II-YI medaka (Oryzias latipes), which have a genetic sex marker that enables the monitoring of germ cells in live individuals by green fluorescent protein (GFP) fluorescence. In a preliminary experiment, we confirmed that the number of germ cells in untreated genetic females was about ten times the number in untreated genetic males at 10 days posthatch. Consequently, sex could be determined by counting the germ cells or measuring the area of GFP fluorescence in gonads. Eggs obtained from olvas-GFP/ST II-YI medaka were nano-injected with ethinylestradiol (EE2) at concentrations of 0, 0.1, 0.5, 2.5, and 5.0 ng/egg and maintained at 25° C until hatching, then fry were similarly maintained until 10 days posthatch. Results indicated that approximately 30% of the genetic males in the 2.5 and 5.0 ng treatments showed increased gonadal area and increased numbers of germ cells at levels close to those of genetic females. Conversely, proliferation of germ cells of genetic females in the 0.5, 2.5, and 5.0 ng treatments was prevented. In the 5.0 ng treatment, there were no significant differences in either germ cell number or fluorescent area between males and females. These results indicated that EE2 injection into eggs causes abnormal development of gonads in both sexes, and that feminization may occur in genetic males. Histological examination of gonads from adult fish showed complete sex-reversal in genetic males in the 0.5, 2.5, and 5.0 ng treatments at almost the same frequencies as at 10 days posthatch, but no reversal in adult genetic females in any treatment. These results show that observation of GFP fluorescence in gonads is available to reveal the effect of EE2. This method could facilitate tests of the potency of endocrine-disrupting chemicals in fish within 10 days posthatch.
Key words: medaka, transgenic, nanoinjection, endocrine disruption
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