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

This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-alpha (ER alpha) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers base d on the known ER alpha DNA sequence in this species, cDNA sequences repres enting most of the protein coding region were obtained from ovary poly A(+) RNA. Using these DNA sequences as probes in Northern blot hybridizations c onfirmed that a single transcript of 4.2 kilobases in poly A(+) RNA could b e detected in liver and ovary RNA. For the quantitative RT-PCR assay an int ernal standard RNA molecule was produced to control for inherent inter-tube differences in amplification efficiency and permit accurate quantification of ERa mRNAs. The quantitative RT-PCR assay proved to be highly specific f or ER alpha mRNA with a detection limit of 6.9 fg, which corresponds to 273 fg ER alpha mRNA/mug total RNA. The quantitative RT-PCR assay was used to measure the levels of ER alpha mRNA in ovaries of rainbow trout at differen t stages of reproductive development. Ovarian ER alpha mRNA expression was found during two distinct periods of reproductive development, in pre-vitel logenic ovaries of fish with ovarian follicle diameters (OFDs) less than or equal to 100 mum and in mid-vitellogenic ovaries with OFDs>1000 mum. ER al pha: mRNA could not be detected in the ovaries of fish with OFDs >100 mum b ut less than or equal to 1000 mum The highest levels of ER alpha mRNA were found in late vitellogenic ovaries of fish with OFDs >2000 mum.