Quantification of rainbow trout (Oncorhynchus mykiss) estrogen receptor-α messenger RNA and its expression in the ovary during the reproductive cycle

Abstract

This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-α (ERα) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers based on the known ERα 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 ERα mRNAs. The quantitative RT-PCR assay proved to be highly specific for ERα mRNA with a detection limit of 6·9 fg, which corresponds to 273 fg ERα mRNA/μg total RNA. The quantitative RT-PCR assay was used to measure the levels of ERα mRNA in ovaries of rainbow trout at different stages of reproductive development. Ovarian ERα mRNA expression was found during two distinct periods of reproductive development, in pre-vitellogenic ovaries of fish with ovarian follicle diameters (OFDs) ≤ 100 μm and in mid-vitellogenic ovaries with OFDs >1000 μm. ERα mRNA could not be detected in the ovaries of fish with OFDs >100μm but ≤ 1000μm. The highest levels of ERα mRNA were found in late vitellogenic ovaries of fish with OFDs >2000 μm.