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Laboratorio de Endocrinología y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, C C 242, Santa Fe, Argentina

(Requests for offprints should be addressed to E H Luque; Email: eluque{at}fbcb.unl.edu.ar)

^* (J Varayoud and J G Ramos contributed equally to this work)

The gene for estrogen receptor (ER) 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 transcription and translation in the rat uterus by ovarian steroid hormones. We examined whether expression of ER mRNA splice isoforms is hormonally regulated in ovariectomized (OVX) and cycling rats. Adult OVX female rats were treated daily with 17-ß estradiol (E₂) (0.05 µg/rat or 5 µg/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 mRNA isoforms are differentially expressed under various physiological conditions, animals were evaluated at proestrus, estrus and diestrus. The ER protein and mRNA were detected by immunohistochemistry and comparative RT-PCR analysis respectively. The presence of ER mRNA isoforms was evaluated using a nested RT-PCR assay. In OVX control rats, ER mRNA and protein levels were high, demonstrating a constitutive expression of the ER gene in the uterus. When animals received P₄ or the high dose of E₂, a significant decrease in both ER mRNA and protein was observed in the uterus. However, when rats were protein was treated with the low dose of E₂, only the ER down-regulated; no changes were observed in ER mRNA expression. In addition to the full-length ER mRNA, OVX control rat uteri expressed three shorter transcripts: 3, 4 and 3,4 (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 3 isoform was completely abolished. During the estrous cycle, all ER mRNA splicing variants were detected at proestrus and estrus. However, in diestrus, significant low levels of the 3 isoform were observed. In summary, our results suggest a dose-dependent relationship between E₂ concentrations and the level of control in the ER transcription–translation cascade. Moreover, the alternative splicing of the ER 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.

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