The ontogeny of nuclear estrogen receptor isoform expression and the effect of 17β-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 ERα isoforms (α1/α2) and the two ERβ isoforms (β1/β2) in male and female embryos from 50 to 600 degree-days (DD; days post-fertilization × water temperature) and in embryos exposed to vehicle or 17β-estradiol (E2) for 2 h 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 ERα2 mRNA was significantly greater in males than in females and at 100 DD the level of ERβ1 mRNA was significantly greater in females than in males ( p < 0.05). Expression profiles of the two ERα isoforms were slightly different from one another, whereas the ERβ isoforms exhibited similar expression patterns. The effect of E2 was not different between male and female embryos. The level of ERα1 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 ERα1 is the only E2 responsive isoform

Variation Among Rainbow Trout (Oncorhynchus mykiss) Estrogen Receptor Isoform 3′ Untranslated Regions and the Effect of 17β-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 17β-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 ERα1 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 ERα1 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 ERα1 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

Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle

Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (β), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erβ1, erβ2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17β, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erβ1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17β levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too