Variation among rainbow trout (Oncorhynchus mykiss) estrogen receptor isoform 3' untranslated regions and the effect of 17beta-estradiol on mRNA stability in hepatocyte culture.

Adenine and uridine (AU)-rich elements in the 3' untranslated region (3'UTR) have been implicated in the 17beta-estradiol (E2) stabilization of vertebrate estrogen receptor (ER) mRNAs. To date, fishes have the most complex arrangement of nuclear ERs with up to two isoforms of each of the two genes in some species (i.e., four different ERs). The objective of this study was to analyze the sequence variation of 3'UTRs among the four ER isoforms in the rainbow trout and determine to what degree it is responsible for the estrogen-induced increase of ER mRNAs in the liver of this fish. This was done by comparing the 3'UTR DNA sequence length and composition, and by measuring expression of ER isoform 3'UTR luciferase reporter constructs in primary cultures of trout hepatocytes treated with E2. There were large differences both in overall length and in sequence composition among the four ER isoform 3'UTRs. The ERalpha1 sequence was the longest and the only one of the four that contained multiple copies of the canonical AU-rich elements (AUUUA) as well as the stability sequence (GCUGAU). E2 treatment significantly increased the luciferase activity in cells transiently transfected with the ERalpha1 reporter construct, relative to cells transfected with reporter vectors containing the other three ER isoform 3'UTRs or the parental vector control. These results support the hypothesis that the E2-induced increase in hepatic ERalpha1 mRNA in rainbow trout is due in part to sequence variability among ER isoform 3'UTRs. We conclude that posttranscriptional stabilization of ER mRNA by E2 appears to be conserved among vertebrates.

The ontogeny of nuclear estrogen receptor isoform expression and the effect of 17beta-estradiol in embryonic rainbow trout (Oncorhynchus mykiss).

Ligand bound nuclear estrogen receptor (ER) acts as a transcription factor regulating the expression of estrogen dependent genes. There are four nuclear ER isoforms in rainbow trout (Oncorhynchus mykiss). The objective of this study was to measure whole body mRNA levels of the two ERalpha isoforms (alpha1/alpha2) and the two ERbeta isoforms (beta1/beta2) in male and female embryos from 50 to 600 degree-days (DD; days post-fertilizationxwater temperature) and in embryos exposed to vehicle or 17beta-estradiol (E2) for 2h at 230, 240 and 250 DD. All four isoforms were detected at every time point in both sexes. Sexual dimorphism was rarely observed; at 50 DD the level of ERalpha2 mRNA was significantly greater in males than in females and at 100 DD the level of ERbeta1 mRNA was significantly greater in females than in males (p<0.05). Expression profiles of the two ERalpha isoforms were slightly different from one another, whereas the ERbeta isoforms exhibited similar expression patterns. The effect of E2 was not different between male and female embryos. The level of ERalpha1 mRNA increased significantly at 240 DD; a similar but not statistically significant trend was observed at 230 and 250 DD. Despite the critical role of estrogen during sex differentiation in rainbow trout, the receptivity to this hormone as measured by the response in mRNA levels of ER appears to be largely the same between males and females and ERalpha1 is the only E2 responsive isoform.

Regulation of hepatic estrogen receptor isoform mRNA expression in rainbow trout (Oncorhynchus mykiss).

The complete nuclear estrogen receptor family in rainbow trout consists of two subtypes (ERalpha and ERbeta) each of which consists of two isoforms (alpha1/alpha2 and beta1/beta2). Transcription rate and mRNA stability of ERalpha1 is affected by 17beta-estradiol (E2) but no information on estrogen regulation exists for the other isoforms. The objective of this study was to compare the mRNA expression patterns of the four ER isoforms in the liver of male trout and in immortalized trout hepatocyte lines (RTH-149 and SOB-15) treated with E2 or 17alpha-ethynylestradiol (EE2) using quantitative RT-PCR. To determine the in vivo dose-response, isogenic male trout were injected intra-peritoneally with 0, 1.5, 15 or 150 microg E2 or an equimolar amount of EE2 and the liver sampled 24 h later. Treatment with either E2 or EE2 significantly (p<0.05) increased the level of ERalpha1 mRNA at all doses tested compared to vehicle, while the response of mRNAs for the other three isoforms did not change. The in vitro dose-response was tested by treating both cell lines with 0, 0.1, 1.0 or 10.0 microM E2 for 48 h. In RTH-149 cells, ERalpha1, ERalpha2 and ERbeta2 mRNAs were significantly higher in cells incubated with 10 microM E2 as compared to cells treated with only vehicle (p<0.05). In SOB-15 cells, ERalpha2 and ERbeta1 mRNAs were significantly higher in cells incubated with 1.0 microM E2 as compared to cells incubated with only vehicle (p<0.05). These results support the conclusion that the mRNAs for the four ER isoforms respond differentially to estrogen regulation.