Detection of environmental estrogenicity using transgenic medaka hatchlings (Oryzias latipes) expressing the GFP-tagged choriogenin L Gene

Abstract

The discharge of environmental estrogenic substances into the environment has an adverse effect on human and wildlife, especially aquatic organisms. Therefore, a simple, practical and sensitive method of detecting environmental estrogenicity is required. Previously, we established a transgenic medaka (Oryzias latipes) strain harboring choriogenin L (ChgL) tagged with green fluorescence protein (GFP), which is expressed in the liver in response to estrogen (E2). This strain of medaka could be a very useful tool in detecting aquatic estrogenicity. The appropriate conditions for analysis of estrogenicity were determined at various E2 concentrations, exposure periods and the developmental stages of medaka hatchlings. Furthermore, the relationship between E2 concentrations and GFP fluorescence intensity was investigated. It was found that fluorescence intensity of GFP depends largely on E2 concentration, exposure time and developmental stage. Hatchling at 4-day post-hatch (DPH) showed optimum conditions for exposure to E2 with optimum GFP intensity at 9 DPH. Additionally, the exposure period was optimized so that exposure from 4 DPH for 5 days showed a significant change in GFP intensity. E2 concentrations of 0, 12.5, 25, 50 and 100 ng/L were used, with 25 ng/L showing a clear increase in GFP intensity at day 6 of exposure. The sensitivity of vitellogenin (Vtg) induction was also examined by Western blot and enzyme-linked immunosorbent assay (ELISA) using whole-body homogenates of E2-exposed (0, 12.5, 25, 50 and 100 ng/L) juvenile medaka. Vitellogenin induction, as determined by Western blot, was found in those juveniles exposed to E2 at a concentration of 100 ng/L. Whereas, Vtg induction was detected by ELISA from juveniles exposed to 12.5 ng/L of E2. The results suggest that ChgL-GFP transgenic medaka could be a simple and practical tool in detecting environmental estrogenicity considering the actual concentrations of estrogenic activity in contaminated and/or wastewater.

Keywords:

• Medaka
• choriogenin
• GFP
• vitellogenin
• estrogen
• endocrine disruptors

Acknowledgments

This work was supported by grants by the Japan Society for the Promotion of Science (JSPS) to MAS as a Post-doc Fellow and SH (1600449), partly by the grants for 2005 Strategic Research Project (No. K17030) of Yokohama City University, Japan and from CREST Project of JPST for “Endocrine Disruption on Action of Brain Neurosteroids.”

This paper was presented at the SETAC Asia/Pacific 2006 conference held at Peking University, China on September 18–20, 2006.