The estrogen receptor α Σ3 mRNA splicing variant is differentially regulated by estrogen and progesterone in the rat uterus

Abstract

The gene for estrogen receptor alpha (ER alpha) has been shown to be under complex hormonal control and its activity can be regulated by mRNA alternative splicing. Here we examined the regulation of ER alpha transcription and translation in the rat uterus by ovarian steroid hormones. We examined whether expression of ER alpha mRNA splice isoforms is hormonally regulated in ovariectomized (OVX) and cycling rats. Adult OVX female rats were treated daily with 17-beta estradiol (E2) (0.05 microg/rat or 5 microg/rat), progesterone (P4) (1 mg/rat) or a combination of both hormones for 4 days. Animals were killed 24 h after the last injection and uterine horns were removed. In order to determine whether ER alpha mRNA isoforms are differentially expressed under various physiological conditions, animals were evaluated at proestrus, estrus and diestrus. The ER alpha protein and mRNA were detected by immunohistochemistry and comparative RT-PCR analysis respectively. The presence of ER alpha mRNA isoforms was evaluated using a nested RT-PCR assay. In OVX control rats, ER alpha mRNA and protein levels were high, demonstrating a constitutive expression of the ER alpha gene in the uterus. When animals received P4 or the high dose of E2, a significant decrease in both ER alpha mRNA and protein was observed in the uterus. However, when rats were protein was treated with the low dose of E2, only the ER alpha down-regulated; no changes were observed in ER alpha mRNA expression. In addition to the full-length ER alpha mRNA, OVX control rat uteri expressed three shorter transcripts: sigma3, sigma4 and sigma3,4 (lacking exon 3, exon 4, or both 3 and 4 respectively). Surprisingly, when OVX animals were treated with P4, the low dose of E2 or a combination of both steroids, expression of the sigma3 isoform was completely abolished. During the estrous cycle, all ER alpha mRNA splicing variants were detected at proestrus and estrus. However, in diestrus, significant low levels of the sigma3 isoform were observed. In summary, our results suggest a dose-dependent relationship between E2 concentrations and the level of control in the ER alpha transcription-translation cascade. Moreover, the alternative splicing of the ER alpha primary transcript is influenced by the hormonal milieu, suggesting that these events could affect the estrogen responsiveness of the rat uterus during the estrous cycle.