TP17 Biomarkers
Tuesday, 15 November 2005: 8:00 AM - 6:30 PM in Exhibit Hall

(PAR-1117-838209) Application of in situ hybridization to detect CYP19A1 and A2 gene expression patterns along the HPG-axis at different organizational levels using Japanese Medaka (Oryzias latipes).

Park, JW¹, Tompsett, AR¹, Hecker, M¹, Jones, PD¹, Newsted, JL², Giesy, JP^{1, 2}, ¹ Zoology Department, Michigan State University, East Lansing, MI, USA² Entrix, Inc., Okemos, MI, USA

ABSTRACT- Many chemicals have the potential to affect endocrine systems of aquatic organisms but until recently, most of the effort to evaluate the effects of endocrine disrupters has focused on steroid receptor mediated effects. Furthermore, the methods to screen for these compounds have been limited by examining only one or a few tissues or only one time during the development of the organism. Because chemicals can cause both direct (receptor-mediated) and indirect effects through changes in signal transduction pathways, it is necessary to simultaneously screen a number of tissues during critical windows of development. Thus, we aimed to develop an in situ hybridization technique to screen for effects of chemicals on the hypothalamic-pituitary-gonadal (HPG) axis with a special emphasis on steroidogenic pathways and hormonal control mechanisms along the HPG-axis in the Japanese medaka. Initially, we selected three genes (CYP19A1, CYP19A2 and beta-actin) to evaluate the proposed test system. The expression of CYP19 genes were studied in that CYP19, aromatase, is involved in the terminal conversion of testosterone to estradiol and has been hypothesized to be involved in the process of feminization of some aquatic organisms. Beta-actin was used as a house keeping gene to standardize the expression levels in the tissues of medaka. Whole medaka fish were longitudinally sectioned, fixed, and embedded in paraffin. Sections were hybridized with each single stranded DNA probe labeled with a fluorescent dye. Fluorescent image data were collected and analyzed quantitatively by determining the intensity and fluorescent areas among the different tissues on each whole animal slide using a Sony CCD camera and the Gel Expert program. It could be demonstrated that the developed test system is a highly sensitive method to determine expression of various genes simultaneously in multiple tissues within an entire organism

Key words: in situ hybridization, CYP19A1 and A2, screening, Japanese medaka