Ligand-binding studies revealed the presence of GnRH-binding sites in African catfish ovary. However, our expression profiling studies failed to detect the previously identified catfish GnRH receptor (cfGnRH-R1) mRNA in this tissue. This negative result instigated us to clone an additional catfish GnRH receptor (cfGnRH-R2) cDNA and study its expression in different tissues in conjunction with the expression of the two catfish GnRH (i.e. cfGnRH and cGnRH-II) genes. The highest cfGnRH-R1 and cfGnRH-R2 mRNA levels were detected in pituitary for cfGnRH-R1 and in brain and ovary for cfGnRH-R2. cfGnRH mRNA was coexpressed with cfGnRH-R1 mRNA in pituitary and brain and with cfGnRH-R2 mRNA in brain and ovary. Ubiquitous expression of cGnRH-II mRNA was observed in all tissues tested, with the highest expression in brain, heart, pituitary, ovary, and head-kidney. Binding studies revealed that cfGnRH-R1 had a higher affinity than cfGnRH-R2 for cGnRH-II, cfGnRH, and various other GnRH agonists. However, this was not reflected in the inositol phosphate or cAMP signal transduction properties of both types of cfGnRH-R. We therefore conclude that in catfish, functional ligand/receptor units evolved by restricted coexpression of a particular receptor in combination with a particular GnRH in particular (nearby) tissue(s).

The gene and cDNA encoding a putative follicle-stimulating hormone beta subunit (cfFSHbeta) from African catfish (Clarias gariepinus) were cloned. Similar to other FSHbeta genes, the cfFSHbeta gene consisted of three exons interrupted by two introns. The cfFSHbeta cDNA coded for a mature protein of 115 amino acids. The 12 cysteines that are required for the typical tertiary folding of glycoprotein hormone beta subunits were positionally conserved in cfFSHbeta. The cfFSHbeta mRNA expression was exclusively detected in the pituitary and was detectable before pubertal development was initiated. The cfFSHbeta transcript levels increased in particular during early stages of puberty and reached constantly high levels after the first appearance of spermatids in the testis. The cfFSHbeta mRNA-positive cells were localized in the proximal pars distalis. Castration of mature males caused elevated cfFSHbeta mRNA levels that were decreased by steroid replacement. Previous work indicated that the African catfish is an interesting model to study the regulation of gonadal functions because cfLH is able to activate both the catfish luteinizing hormone receptor (cfLH-R) and follicle-stimulating hormone receptor (cfFSH-R). Because cfFSH purification has failed so far, ongoing studies are directed toward the production of recombinant cfFSH. After all, the developmental and hormonal regulation of cfFSHbeta transcript levels opens the possibility for physiologically relevant actions of the putative cfFSH, next to the presumptive bifunctionally acting cfLH.

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.