PACAP system evolution and its role in melanophore function in teleost fish skin.

Pituitary adenylate cyclase-activating polypeptide (PACAP) administered to tilapia melanophores ex-vivo causes significant pigment aggregation and this is a newly identified function for this peptide in fish. The G-protein coupled receptors (GPCRs), adcyap1r1a (encoding Pac1a) and vipr2a (encoding Vpac2a), are the only receptors in melanophores with appreciable levels of expression and are significantly (p < 0.05) down-regulated in the absence of light. Vpac2a is activated exclusively by peptide histidine isoleucine (PHI), which suggests that Pac1a mediates the melanin aggregating effect of PACAP on melanophores. Paradoxically activation of Pac1a with PACAP caused a rise in cAMP, which in fish melanophores is associated with melanin dispersion. We hypothesise that the duplicate adcyap1ra and vipr2a genes in teleosts have acquired a specific role in skin and that the melanin aggregating effect of PACAP results from the interaction of Pac1a with Ramp that attenuates cAMP-dependent PKA activity and favours the Ca(2+)/Calmodulin dependent pathway.

Novel galanin receptors in teleost fish: identification, expression and regulation by sex steroids.

In fish, the onset of puberty, the transition from juvenile to sexually reproductive adult animals, is triggered by the activation of pituitary gonadotropin secretion and its timing is influenced by external and internal factors that include the growth/adiposity status of the animal. Kisspeptins have been implicated in the activation of puberty but peripheral signals coming from the immature gonad or associated to the metabolic/nutritional status are also thought to be involved. Therefore we hypothesize the importance of the galinergic system in the brain and testis of pre-pubertal male sea bass as a candidate to translate the signals leading to activation of testicular maturation. Here, the transcripts for four galanin receptors (GALR), named GALR1a, 1b, 2a and 2b, were isolated from European sea bass, Dicentrarchus labrax. Phylogenetic analysis confirmed the previously reported duplication of GALR1 in teleost fish, and unravelled the duplication of GALR2 in teleost fish and in some tetrapod species. Comparison with human showed that the key amino acids involved in ligand binding are present in the corresponding GALR1 and GALR2 orthologs. Transcripts for all four receptors are expressed in brain and testes of adult fish with GALR1a and GALR1b abundant in testes and hardly detected in ovaries. In order to investigate whether GALR1 dimorphic expression was dependent on steroid context we evaluated the effect of 11-ketotestosterone and 17ß-estradiol treatments on the receptor expression in brain and testes of pre-pubertal males. Interestingly, steroid treatments had no effect on the expression of GALRs in the brain while in the testes, GALR1a and GALR1b were significantly up regulated by 11KT. Altogether, these results support a role for the galaninergic system, in particular the GALR1 paralog, in fish reproductive function.

DAX1 regulatory networks unveil conserved and potentially new functions.

DAX1 is an orphan nuclear receptor with actions in mammalian sex determination, regulation of steroidogenesis, embryonic development and neural differentiation. Conserved patterns of DAX1 gene expression from mammals to fish have been taken to suggest conserved function. In the present study, the European sea bass, Dicentrarchus labrax, DAX1 promoter was isolated and its conserved features compared to other fish and mammalian DAX1 promoters in order to derive common regulators and functional gene networks. Fish and mammalian DAX1 promoters share common sets of transcription factor frameworks which were also present in the promoter region of another 127 genes. Pathway analysis clustered these into candidate gene networks associated with the fish and mammalian DAX1. The networks identified are concordant with described functions for DAX1 in embryogenesis, regulation of transcription, endocrine development and steroid production. Novel candidate gene network partners were also identified, which implicate DAX1 in ion homeostasis and transport, lipid transport and skeletal development. Experimental evidence is provided supporting roles for DAX1 in steroid signalling and osmoregulation in fish. These results highlight the usefulness of the in silico comparative approach to analyse gene regulation for hypothesis generation. Conserved promoter architecture can be used also to predict potentially new gene functions. The approach reported can be applied to genes from model and non-model species.

Ca(2+)-Calmodulin regulation of testicular androgen production in Mozambique tilapia (Oreochromis mossambicus).

The Ca(2+)-Calmodulin (CaM) signaling pathway has previously been shown to be involved in the regulation of teleost fish ovarian steroidogenesis. However, a putative role of CaM in testicular steroidogenesis and potential targets has not been examined. To examine whether basal steroidogenesis is modulated by Ca(2+) and CaM levels in the testis of Mozambique tilapia (Oreochromis mossambicus) we have incubated testicular fragments in vitro under different conditions and analyzed steroid output. Calcium-free medium with or without EGTA did not affect testicular basal 11-ketotestosterone (11-KT) and testosterone (T) secretion. However, addition of 80microM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80microM of W7 was accompanied by increased 11-desoxicortisol output and by the activation of cortisol synthesis in the testis, the latter undetected in untreated tissues. However, production of 17,20alpha-dihydroxy-4-pregnen-3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11beta-hydroxysteroid dehydrogenase are targets for CaM. In addition, androgen production was also found to be regulated by the level of cAMP since incubations with forskolin (FK) significantly increased 11-KT and T output. A cross-talk between the cAMP and Ca(2+)-CaM signaling pathways was detected since W7 administration also decreased FK stimulated androgen production. Altogether, these data show that both basal and cAMP stimulated androgen levels were modulated by intracellular Ca(2+)-dependent CaM and that possibly Ca(2+)-CaM determines the shift in steroidogenesis from C21 steroids to androgens.