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Chemical communication is widespread in the animal kingdom and olfaction constitutes a powerful channel for social and environmental cues. In fish, olfactory stimuli are known to influence physiological processes, including reproduction. Here we investigate the effects of olfaction on puberty in European sea bass Dicentrarchus labrax males. Intact sea bass coming to first maturity (puberty) are able to smell conspecific odours. However, induced anosmia during most of the spermatogenesis period had no effect on the sex ratio, gonad maturation state or gonado-somatic index at the time of reproduction. Furthermore anosmia decreased mRNA expression of brain KISS2 and pituitary LHb and FSHb, but not brain GnRH1 and GnRH3. Thus, although anosmia seems to modify gene expression of key reproduction related genetic factors, it seems to be insufficient to stop or delay growth or gonadal development and maturation.

In the present study the calcitonin receptor (CTR) sub-family of family B G-protein coupled receptors (GPCRs) in teleosts is evaluated and put in the context of the families overall evolution from echinodermates to vertebrates. Echinodermates, hemichordates, cephalochordates and tunicates have a single gene that encodes a receptor that bears similarity to the vertebrate calcitonin receptor (CTR) and calcitoninlike receptor (CTR/CLR). In tetrapods one gene encodes the calcitonin receptor (CALCR) and another gene the calcitonin receptor-like receptor (CALCRL). The evolution of CALCR has been under strong conservative pressure and a single copy is also found in fishes and high conservation of gene organisation and synteny exits from teleosts to human. A teleost specific CTR innovation that occurred after their divergence from holostei is the presence of several HBDs in the N-terminus. CALCRL had a different evolutionary trajectory from CALCR and although a single gene copy is present in tetrapods the sarcopterygii fish, the coelacanth, has 1 copy of CALCRL but also a fish specific form CALCRL3. The ray-finned fish, the spotted gar, has 1 copy of CALCRL and 1 of CALCRL3 but the teleost specific whole genome duplication has resulted in a CALCRL1 and CALCRL2 in addition to the fish specific CALCRL3. Strong conservation of CALCRL gene structure exists from human to fish. Promoter analysis in silico reveals that the duplicated CALCRL genes in the teleosts, zebrafish, takifugu, tetraodon and medaka, have divergent promoters and different putative co-regulated gene partners suggesting their function is different. We are grateful to Professor Adelino Canário for providing the sequence of sea bass EST clones. This study was supported by the European Regional Development Fund (ERDF) COMPETE – Operational Competitiveness Programme and Portuguese funds through FCT – Foundation for Science and Technology, under the project ‘‘PEst-C/MAR/LA0015/2013’’ and by FCT PTDC/BIA-BCM//73597/ 2010. RM (SFRH/BPD/66742/2009) and FV (SFRH/BPD/73597/ 2010) were in receipt of a post-doctoral grant from FCT, Portugal.

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.