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MEASUREMENT OF ESTROGEN-INDUCED VITELLOGENIN AND VITELLINE ENVELOPE PROTEIN MRNA IN SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS) USING QUANTITATIVE REAL-TIME PCR TECHNOLOGY
Citation:
Knoebl, I., M J. Hemmer, AND N D. Denslow. MEASUREMENT OF ESTROGEN-INDUCED VITELLOGENIN AND VITELLINE ENVELOPE PROTEIN MRNA IN SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS) USING QUANTITATIVE REAL-TIME PCR TECHNOLOGY. Presented at SETAC Europe 13th Annual Meeting, Hamburg, Germany, Apr 27 - May 1, 2003.
Description:
Many environmentally persistent chemicals found in both European and U.S. waterways can act as estrogens by binding to estrogen receptors and modifying the expression of genes regulated by endogenous estrogens. Synthesis of female-specific proteins (Vitellogenin [VTG], vitelline envelope proteins [ZP]) in male fish is used as an indicator of estrogen exposure. However, protein synthesis can be delayed up to several days after exposure to estrogenic chemicals. An indicator of more immediate changes in gene expression is measurement of changes in mRNA levels. Microarray technology was developed to assess changes in gene expression in male sheepshead minnows exposed to varying concentrations of 17b-estradiol and nonylphenol. Quantitative real-time PCR assays were also developed to measure the hepatic mRNA of two VTGs (a and b) and two ZPs (2 and 3). Using this technology, increases in both VTG a and b were observed in male fish exposed for 6 h to 100 ng/L of estradiol. Male sheepshead minnows were exposed to five concentrations of 17b-estradiol (50, 100, 200, 500 and 1000 ng/L) for various times (6, 12, 24, 48 h and 2 to 22 d). Dose-response changes in VTG mRNA levels were found with 10-fold lower concentrations of VTG b compared to VTG a. Male fish were also exposed to concentrations of nonlyphenol ranging from 0.64 to 42.7 ug/L for 2 to 56 days. Temporal and dose-response comparisons of mRNA induction for two VTGs and two ZPs were measured by quantitative real-time PCR. Changes in expression of other genes of interest using microarray technology are also presented.