Environ. Sci. Technol. 2007, 41, 1473-1479
Quantitative Bioimaging Analysis of Gonads in olvas-GFP/ST-II YI Medaka (Transgenic Oryzias latipes) Exposed to Ethinylestradiol T A K E S H I H A N O , † Y U J I O S H I M A , * ,† MASATO KINOSHITA,‡ MINORU TANAKA,§ NORIKO MISHIMA,| TETSUJI OHYAMA,⊥ TAKASHI YANAGAWA,⊥ YUKO WAKAMATSU,| KENJIRO OZATO,| AND TSUNEO HONJO† Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita 10-jyo, Nishi 8-chome, Kita-ku, Sapporo 060-0810, Japan, Laboratory of Freshwater Fish Stocks, Bioscience and Biotechnology Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, and Institute of Biostatistics, Kurume University Graduate School of Medicine, Asahi-machi, Kurume, 830-0011, Japan
This study investigates the adverse and persistent effects of ethinylestradiol (EE2) on mature gonads of transgenic olvas-GFP/ST II-YI medaka (Oryzias latipes). The measurement of gonadal size calculating the GFP-fluorescent area was used as a technique that enabled monitoring gonads in living specimens by GFP fluorescence. First, mature medaka were exposed to EE2 (47.8-522 ng/L) for 4 weeks. The gonads showed a significant reduction of the GFPfluorescent area and Gonadosomatic Index in males exposed to EE2 at >216 ng/L and females exposed at 522 ng/L. Histologically, males at all treatments exhibited testis-ova and additionally, high connective tissue prevalence at g216 ng/L. Next, mature male medaka were exposed to EE2 (43.7-473 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 g215 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. Alterations in GFP fluorescence clearly indicate the condition of the gonad in transgenic medaka and this strain showed a facilitated screening fish model to detect the adverse effects on the gonad by estrogenic chemicals.
* Corresponding author telephone: +81-92-642-2905; fax: +8192-642-2908; e-mail:
[email protected]. † Kyushu University. ‡ Kyoto University. § Hokkaido University. | Nagoya University. ⊥ Kurume University Graduate School of Medicine. 10.1021/es0620134 CCC: $37.00 Published on Web 01/19/2007
2007 American Chemical Society
Introduction In the past few years, general concerns about the release of estrogens into the environment have increased because of their potential to cause adverse physiological effects in wildlife. Chemicals possessing estrogenic activity range from synthetic compounds such as 17R-ethinylestradiol (EE2), nonylphenol, and bisphenol A to natural estrogens such as 17β-estradiol (E2) and phytoestrogens. Mixtures of xenoestrogens in the aquatic environment may interfere with sexual development and reproduction in fish and other animals. In field studies, the reported effects of exposure to estrogen-mimicking chemicals include induction of intersex gonads in roaches (Rutilus rutilus) collected near sewage treatment plants (1) and hypertrophy of connective tissues in male flounders collected from estuary (2). EE2 is used in contraceptive formulations because of its strong estrogenic potency (3) and has been detected in many sewage treatment work effluents and in many surface waters. The concentrations of EE2 in river surface waters were relatively low and reported to be a few nanograms per liter (4). In laboratory experiments, EE2 induces intersex gonad in medaka (Oryzias latipes) at 0.1 ng/L (5) and decreases fertility in sexually maturing male rainbow trout (Oncorhynchus mykiss) at 10 ng/L (6) and in zebrafish (Danio rerio) at 3 ng/L (7). Hence, EE2 is suspected to be one of the causes of estrogenic effects observed in wild fish (8). Despite the frequently low environmental concentrations of EE2, one exceptional high levels of EE2 concentrations to these lower values is a recent survey of U.S. waters that reported EE2 concentrations ranging from 73 to 831 ng/L (9). These high levels of concentrations were critical to aquatic organisms, previously reported as impairment of reproductive capabilities in medaka exposed to 488 ng/L of EE2 for 3 weeks (10). Impairment of reproductive capacity derives from decreases in fertility, and lesioned gonad by estrogenic chemicals, in which abnormal connective tissues was developed, was found to be causative (10, 11). Considering that abnormal changes in fish testicular structure have been found in the aquatic environment (2), it is important to develop a facilitated system to monitor the gonadal change adversely affected by temporarily high levels of estrogenic chemicals released into the environment. In addition, although many studies have focused on the effect of estrogenic compounds on the testis, little is known whether testis lesions and impaired spermatogenesis induced by estrogenic chemicals can be reconstituted in adult medaka transferred to clean water. Transgenic fish are useful not only for unraveling the mechanisms of sexual differentiation, but also for detecting the effects of chemicals in in vivo experiments. The olvasGFP/ST-II YI strain is an ideal model to monitor the gonad in a living organism, by which expression of green fluorescent protein (GFP) can be visualized. This strain was produced from see-through medaka (12) by fusing the gene for GFP to the regulatory region of the medaka vasa (olvas) gene that shows germ line-specific expression during embryonic development and throughout the medaka lifespan (13). Previously, Hano et al. (14) reported that EE2 nanoinjected into olvas-GFP/ST-II YI medaka eggs produces both abnormal sexual differentiation at 10 dph in juveniles of both sexes and at 100 dph in adult males, as detected by GFP fluorescence. Kurauchi et al. (15) indicated that the choriogenin H (CHgH) -GFP transgenic medaka strain could be used as an alternative method to detect the estrogenic activity in environmental water samples. VOL. 41, NO. 4, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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In this study, we examined the effects of different concentrations of EE2 on matured gonads of living medaka specimens by GFP fluorescence and the recovery of GFPfluorescent area in testis after the exposed males were transferred to clean water. The purpose of this study was to evaluate the application of olvas-GFP/ST-II YI strain to facilitated screening fish model to detect the adverse effects on the gonad by estrogenic chemicals.
Materials and Methods Test Organism. Olvas-GFP/STII YI medaka were maintained at Nagoya University. Medaka selected for this study were approximately 2 month posthatch and were fully mature (body weight 334 ( 66 mg; total length 33 ( 2 mm). They were kept in 16-L tanks with a water circulating system (MH; Meito-suien, Nagoya, Japan) at 26 °C under a 14-h light and 10-h dark cycle, and fed three times daily ad libitum with Artemia nauplii (
