Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)

June-Woo PARK, Amber TOMPSETT, Xiaowei ZHANG, John L. NEWSTED, Paul D. JONES, Doris AU, Richard KONG, Shiu Sun Rudolf WU, John P. GIESY, Markus HECKER

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Abstract

The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 μg/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells. Copyright © 2008 Elsevier Inc. All rights reserved.

Original languageEnglish
Pages (from-to)226-235
JournalToxicology and Applied Pharmacology
Volume232
Issue number2
DOIs
Publication statusPublished - 15 Oct 2008

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Oryzias
Fluorescence In Situ Hybridization
Gene expression
Fluorescence
Tissue
Gene Expression
Fadrozole
Messenger RNA
In Situ Hybridization
Genes
Polymerase Chain Reaction
Fixatives
Aromatase Inhibitors
Histology
Aromatase
Confocal microscopy
Fluorescence microscopy
Gonads
Transcriptome
Fluorescence Microscopy

Citation

Park, J.-W., Tompsett, A., Zhang, X., Newsted, J. L., Jones, P. D., Au, D., . . . Hecker, M. (2008). Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes). Toxicology and Applied Pharmacology, 232(2), 226-235. doi: 10.1016/j.taap.2008.06.012

Keywords

  • CYP19a
  • Fadrozole
  • Autofluorescence
  • Histology
  • Endocrine disruptors
  • Fish
  • Aromatase