Molecular cloning, gene expression and characterization of the third estrogen receptor of the Nile tilapia, Oreochromis niloticus

Abstract

Estrogens are essential for many reproductive and non-reproductive functions. In teleosts, it is well-known that several subtypes of estrogen receptors are required for the precise action of estrogens. Present study describes the cloning of the third estrogen receptor, ER- beta2, from the Nile tilapia by EST sequencing coupled microarray. The cloned ER-beta2 showed 77.7% amino acid identity with the reported Atlantic croaker ER-beta. Three ERs, ER-alpha, ER-beta1 and ER-beta2, from the fugu genome were also isolated to analyze their gene structures. Comparison of the intron/exon boundaries and exon numbers of fugu, tilapia, rainbow trout and zebrafish, and phylogenetic analysis of 63 ER sequences revealed that ER-beta probably underwent two successive lineage-specific duplications in teleost. The former took place only in zebrafish lineage, and the latter took place in advanced teleosts without the zebrafish lineage, whereas no duplication of the ER-alpha gene has been detected. Tissue distribution analysis by RT-PCR revealed that tilapia ER-alpha and ER-beta1 were expressed ubiquitously, whereas ER-beta2 is expressed only in the pituitary, liver, intestine, kidney and gonads, with the highest expression in the testis and the lowest level in the ovary. Northern blot analysis detected a single transcript of about 3.4 kb in the testis but not in the ovary mRNAs. In transient transfection assays using human embryonic kidney 293 (HEK293) cells, tilapia ER-beta2 showed estrodiol-17beta dependent transactivation.