Immunohistochemical localization and biochemical characterization of two novel decapeptides derived from POMC-A in the trout hypothalamus.

Several vertebrate species which underwent duplication of their genome, such as trout, salmon and Xenopus, possess two proopiomelanocortin (POMC) genes. In the trout, one of the POMC molecules, called POMC-A, exhibits a unique C-terminal extension of 25 amino acids which has no equivalent in other POMCs characterized so far. This C-terminal peptide contains three pairs of basic residues, suggesting that it may be the source of novel regulatory peptides. The aim of the present study was to investigate the occurrence of these peptides in the brain of the trout Oncorhynchus mykiss by using specific antibodies raised against two epitopes derived from the C-terminal extension of POMC-A, i.e., EQWGREEGEE and YHFQ-NH2. Immunohistochemical labeling of brain sections revealed the presence of EQWGREEGEE- and YHFQ-NH2-immunoreactive cell bodies in the anterior part of the nucleus lateralis tuberis of the hypothalamus. Immunoreactive fibers were observed in the dorsal hypothalamus, the thalamus, the telencephalon, the optic tectum and the medulla oblongata. In contrast, no labeling was detected using antibodies against the non-amidated peptide YHFQG. Biochemical characterization was performed by combining high-performance liquid chromatography (HPLC) analysis with radioimmunoassay (RIA) quantification. Two peptides exhibiting the same retention time as synthetic EQWGREEGEE and ALGERKYHFQ-NH2 were resolved. However, no peptide co-eluting with YHFQ-NH2 or YHFQG could be detected. These results demonstrate that, in the trout brain, post-translational processing of POMC-A generates the two decapeptides EQWGREEGEE and ALGERKYHFQ-NH2. The wide distribution of immunoreactive fibers in the diencephalon, telencephalon, optic tectum and medulla oblongata suggests that these peptides may exert neurotransmitter and/or neuromodulator activities.

Distribution and expression of glucocorticoid receptor mRNA in the forebrain of the rainbow trout.

The expression and distribution of glucocorticoid receptor mRNA was studied in the forebrain of mature female and immature undifferentiated rainbow trout (Oncorhynchus mykiss) by means of Northern blotting and in situ hybridization. A single mRNA species of 7.5 kb was detected in mRNA polyA+ prepared from the anterior brain. In situ hybridization was carried out using a 35S-labelled riboprobe corresponding to the A/B-domain (between nucleotides 1224 and 1763) of the recently cloned rainbow trout glucocorticoid receptor cDNA. Comparison of adjacent sections hybridized with the sense and antisense probes allowed detection of a specific signal with a similar distribution pattern in all animals studied. In the telencephalon, a specific hybridization was detected in scattered cells of the dorsal telencephalic hemisphere, but the stronger signal was consistently observed in the dorsal nucleus, and to a lesser degree in the ventral nucleus of the ventral telencephalon. Heavy hybridization staining was consistently observed in all subdivisions of the preoptic nucleus and the nucleus lateralis tuberis, which are the main hypophysiotrophic regions in fish. A weaker signal was detected in the nucleus anterioris periventricularis, nucleus suprachiasmaticus and thalamic region. The presence of a strong signal in virtually all magnocellular neurons of the nucleus preopticus, known for producing vasotocin, isotocin and corticotropin-releasing factor favors a direct function of cortisol in regulating its own secretion.