For some teleosts, a role has been established for melanin-concentrating hormone (MCH) background adaptation and stress response. !n teleost fishes, prepro-MCH (ppMCH) mRNA is expressed in the hypothalamus, predominantly in neurons of the nucleus lateralis tuberis (NLT) and in scattered cells of the nucleus recessus lateralis (NRL). The response of mature tilapia to different environmental challenges was studied by assessing ppMCH mRNA levels in these two hypothalamic nuclei by quantitative dot blot analysis. Changes in background colour induced pronounced differences in ppMCH mRNA expression in the NLT, but not In the NRL. The NLT of tilapia adapted to a white background contained 2.5 to 3 times more ppMCH mRNA than the NLT of black-adapted fish. The NLT of fish kept on neutral background contained intermediate levels of ppMCH mRNA, which were significantly lower than the levels in white-adapted fish. Oral administration of dexamethasone lowered plasma cortisol concentrations, but had no effect on ppMCH mRNA levels in white-and black-adapted fish. In tilapia exposed to strongly acidified water (pH 3.5), plasma cortisol and ACTH concentrations were highly elevated, and plasma chloride concentrations considerably lower than in controls. These fish responded with a 70% rise in ppMCH mRNA levels in the NLT, which is most probably associated with a stress response evoked by inadequate osmoregulation. After exposure to a milder acidification (pH 4.0) or to seawater no significant changes in ppMCH mRNA levels occurred in either the NLT or the NRL, nor in plasma chloride, cortisol and ACTH levels. A specific increase of ppMCH mRNA levels in the NRL was observed in repeatedly disturbed tilapia. We conclude that MCH neurons in the NLT and NRL of this teleost differentially respond to background colour, acidification and disturbance stress, and that this response is not strictly associated with changes in plasma ions and activity of the pituitary-interrenal axis.

M elanin-concentrating horm one (M CH) is a neuro peptide involved in background adaptation in teleost fish, and in m ultiple regulatory functions in mammals and fish. To study the expression of the M C H preprohorm one (ppM C H ) in teleosts, we first cloned a hypothalamic cD N A encoding the com plete ppM C H of tilapia (Oreochromis mossambicus), and a cRNA probe derived from a 270 bp ppM C H cD N A fragment was used for the expression studies. T he level of ppM C H m R N A expression in tilapia hypothalamus, m easured by dot blot analy sis, was significantly higher in fish adapted to a white background than in black-adapted animals, which is in accordance with the reported M CH plasma and tissue concentrations in fish. N orthern blot analysis not only revealed a strong ppM C H mRNA signal in the hypothalam us, but also the presence of ppM C H m R N A in the neurointerm edi ate lobe (N IL) of the pituitary. In situ hybridization and immunocytochemistry showed that ppM C H mRNA as well as M C H immunoreactivity are located in perikarya of two hypothalamic regions, namely in the nucleus lateralis tuberis (N L T ) and the nucleus recessus lateralis (NRL). Quantitative analysis by dot blot hybridization revealed about eight times more ppM C H m R N A in the N L T than in the N R L and N IL of mature tilapias. ppM C H m RNA in the N IL couLd be localized to cell bodies of the neurohypophysis, which were also M CH immunoreactive.

To determine the hybridization conditions for library screening,Published by Elsevier Science Publishers B. V.

No abstract

Melanin-concentrating hormone (MCH) is a neuroendocrine peptide involved in the regulation of skin pigmentation in teleosts. We isolated and sequenced a 543 bp hypothalamic cDNA encoding the MCH-preprohormone of tilapia (Oreochromis mossambicus). Initially, polymerase chain reaction (PCR) experiments were performed on hypothalamic RNA with a synthetic oligonucleotide primer corresponding to a conserved region of salmon and mammalian MCH peptide and an oligo dT primer. A 0.2 kb PCR fragment was obtained and found to have low but significant nucleotide sequence similarity with the 3' ends of known MCH-mRNAs. Subsequently, the PCR fragment was used to screen λZAP cDNA libraries constructed from tilapia hypothalamic poly(A(+)) RNA. The cloned tilapia MCH preprohormone cDNA encodes a 133-amino acid protein of which 17 amino acids belong to the signal peptide. The MCH peptide sequence is located at the carboxy terminus of the preprohormone structure and is preceded by a pair of arginine residues which can serve as a proteolytic cleavage site. 23 to 25 amino acids further upstream in the prohormone structure three consecutive basic residues are present. Cleavage at this site would yield a 22-amino acid MCH gene-related peptide (Mgrp), which is much larger than (12- to 13-amino acid) salmon and mammalian Mgrp. A comparative structural analysis between tilapia preproMCH and its salmon and mammalian counterparts revealed that the MCH peptide sequence is very well conserved (100% identity with salmon and 75% identity with both rat and human MCH). In contrast, the remaining parts of the preproMCH structures have diverged considerably. Northern blot analysis revealed the presence of tilapia preproMCH mRNA in the hypothalamus and not in other brain regions nor in several peripheral tissues.