This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-(ER ) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers based on the known ER DNA sequence in this species, cDNA sequences representing most of the protein coding region were obtained from ovary poly A(+) RNA. Using these DNA sequences as probes in Northern blot hybridizations confirmed that a single transcript of 4·2 kilobases in poly A(+) RNA could be detected in liver and ovary RNA. For the quantitative RT-PCR assay an internal standard RNA molecule was produced to control for inherent inter-tube differences in amplification efficiency and permit accurate quantification of ER mRNAs. The quantitative RT-PCR assay proved to be highly specific for ER mRNA with a detection limit of 6·9 fg, which corresponds to 273 fg ER mRNA/µg total RNA. The quantitative RT-PCR assay was used to measure the levels of ER mRNA in ovaries of rainbow trout at different stages of reproductive development. Ovarian ER mRNA expression was found during two distinct periods of reproductive development, in pre-vitellogenic ovaries of fish with ovarian follicle diameters (OFDs) c100 µm and in mid-vitellogenic ovaries with OFDs >1000 µm. ER mRNA could not be detected in the ovaries of fish with OFDs >100 µm but c1000 µm. The highest levels of ER mRNA were found in late vitellogenic ovaries of fish with OFDs >2000 µm.
Commonly used indices of fish thyroidal status are based on thyroxine (T4) secretion by thyroid tissue under control of the central brain-pituitary-thyroid axis. However, much of the control of the fish thyroid system also occurs in peripheral tissues, such as liver, by regulating T4 prohormone conversion to biologically active 3,5,3'- triiodothyronine (T3) or to biologically inactive 3,3',5'-triiodothyronine and by regulating T3 conversion to inactive 3,3'-diiodothyronine. These extrathyroidal conversions depend on a family of independently-regulated selenocysteine-containing microsomal deiodinases. We describe deiodination assays and evaluate their potential as biomarkers for exposure to chemicals that directly or indirectly disrupt thyroid hormone homeostasis or thyroidal status. We conclude that deiodination be included in a minimum suite of assays to detect xenobiotic effects on the fish thyroid system.
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