Cloning and expression of a functional estrogen receptor-α from African catfish (Clarias gariepinus) pituitary

Abstract

An African catfish (Clarias gariepinus) estrogen receptor-α (cfERα) cDNA fragment was amplified by RT-PCR, in combination with a modified 3′-RACE procedure, on total RNA extracted from pituitary. This cDNA fragment was used to screen an African catfish pituitary cDNA library. A clone was obtained that contained an open-reading frame coding for a 620 amino acid cfERα protein with a deduced molecular mass of 68.1 kDa. In addition, a partial African catfish estrogen receptor-β (cfERβ) cDNA fragment was amplified by RT-PCR on total RNA extracted from testis. Neighbor-joining analysis was used to infer a phylogenetic classification for cfERα and cfERβ. The tree obtained indicated that there are two major clusters of vertebrate ERs: ERα and ERβ. Within each cluster, teleost and tetrapod ER sister clades could be distinguished. The cfERα clustered with other teleost ERαs, whereas cfERβ clustered with other teleost ERβs. The ligand-induced transcriptional activity of cfERα was demonstrated in a transient gene expression assay using cells in which an acute estrogenic response was created by co-transfecting cultures with recombinant cfERα cDNA expression vector constructs in the presence of an estrogen-dependent reporter plasmid. Real-time, quantitative PCR revealed that cfERα transcripts were most abundantly expressed in pituitary, while in all other tissues tested the relative cfERα mRNA levels were less than ∼5% of the level obtained in pituitary. Moreover, we found that, during pubertal development, the relative cfERα mRNA levels gradually increased in African catfish pituitary.