In mammals, corticotropin-releasing hormone (CRH) is the primary hypothalamic factor mediating stress-induced adrenocorticotropin (ACTH) secretion from the anterior pituitary. However, there are limited studies of this neuroendocrine peptide in non-mammalian vertebrates. In euryhaline fish, CRH may have an integrative role in the control of reproductive, osmoregulatory and nutritional systems. The caudal neurosecretory system (CNSS) is a neuroendocrine system distinct from brain and has been shown to be responsible for secretion of two major neuropeptides, urotensin I and II. Here we report cloning and sites of expression of CRH from the CNSS of the euryhaline flounder.

The CRH precursor cDNA was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and screening of a CNSS cDNA library. The CNSSs were isolated from humanely killed fish. RNA was extracted using the guanidine thiocyanate method and utilized for both RT-PCR and library construction. RT-PCR was carried out using degenerate oligonucleotide primers based on the conserved amino acid sequence of CRH from a range of species. A CNSS cDNA library was constructed into the phage vector l{special}TriplEx2 and screened at high stringency using a specific CRH probe obtained from degenerate RT-PCR. The CRH precursor consists of 168 amino acid residues and the carboxyl terminus represents the 41-amino-acid mature peptide, preceded by Arg-Arg and followed by Gly-Lys as putative cleavage sites. The deduced amino acid sequences of CRF and UI peptides from the same fish exhibit a sequence identity of 51.2 %. The genomic organisation, determined by PCR using specific primers, shows a single intron in the 5â-UTR region, and that the whole CRH coding sequence is contained in a single exon. Using conditions that gave no cross-hybridisation with other genes, Northern blot analysis of a range of flounder tissues confirmed that the CNSS is the major site of expression of CRH gene and also shows the possibility of multiple polyadenylation signals in the 3â untranslated region. RT-PCR using specific primers indicated the presence of CRH transcripts in other tissues, such as brain, optic nerve, spinal cord, gill, head kidney, kidney, intestine and rectum. The primary structure of CRH shows a close similarity between flounder, tilapia and catfish, with more variation compared to orthologous vertebrate and other teleost CRHs. Whether these structural differences reflect the varied and contrasting functions of CRH remains to be investigated.

This work was supported by a grant from the BBSRC.