The sexual dimorphism in dlGALR a and dlGALR b receptors

The sexual dimorphism in dlGALR1a and dlGALR1b receptors found in the gonads of adult fish led us to hypothesize that the gene expression difference between female and males might be attributed to the differences in steroid production between female and male gonads. Interestingly we have found that the expression of dlGALRs in the European sea bass atp citrate lyase inhibitor was not affected by sex steroids, although we cannot discard the possibility that contrasting regional changes in dlGALR transcription may have occurred as we analyzed whole brain extracts. Furthermore, it is possible that the lack of responsiveness of dlGALRs in the brain of male pre-pubertal sea bass may reflect the immaturity of the HPG axis at this stage in the individuals analyzed. However, we did find a significant stimulatory effect of 11KT on dlGALR1a and dlGALR1b gene transcription in the testis. In sea bass males, the levels of T and 11KT in the plasma are very low during the first year of development (Rodrıguez et al., 2000) and at the onset of gametogenesis the levels of 11KT are elevated to support the initiation of spermiogenesis and testicular maturation (Rodrıguez et al., 2004). In the present study, the levels of 11KT in the plasma of control fish are within the reported values for pre-pubertal male sea bass (Rodrıguez et al., 2000, Rodrı́guez et al., 2001) and the administration of 0.5mg/kg 11KT elicited an increase in plasma steroid levels comparable to those of males going through first spawning (Rodrıguez et al., 2000). Thus, it is interesting that at physiological levels, 11KT was able to stimulate the transcription of both dlGALR1a and dlGALR1b in the immature testis. The mechanisms mediating this induction remain to be investigated but may include (a) a direct induction of dlGALR1a and dlGALR1b gene expression in testis by 11KT at putative androgen-response elements; (b) an indirect response to 11KT induction of GAL synthesis or release from fish pituitary (where GAL protein and transcripts have previously been detected (Mensah et al., 2010) or (c) a paracrine response to local stimulation of GAL at the testis (where expression of preprogalanin forms have been detected in goldfish (Unniappan et al., 2003). Future studies to characterize galanin transcripts in sea bass, their patterns of transcript and protein expression in pituitary and testis and their possible response to 11KT treatments, as well as promoter analyses of GAL and GALR genes, would be interesting to investigate these hypotheses. The significance of the 11KT induction of GALR1 transcripts in sea bass immature testis and sex dimorphic expression in adults will require further investigation, although it is possible that the sea bass galaninergic system may have a role in the testicular germ cell differentiation. Indeed, upregulation of GAL expression is correlated to the pluripotency of human embryonic stem cells (ESC), as described for the ESC markers NANOG and SOX2 (Assou et al., 2007), and in mouse Leydig cells, GALR1 expression is inversely correlated to the stage of differentiation of these testicular cells (Ohta et al., 2002). The human GAL gene promoter contains conserved estrogen responsive elements (EREs) half sites that are able to be regulated by E2 (Kofler et al., 1995, Howard et al., 1997) and there are several studies focusing on the effect of E2 in the expression and/or secretion of GAL in either the brain or pituitary of males (Kaplan et al., 1988, Selvais et al., 1995). Although, in the present study, we did not find significant effects of E2 on dlGALRs expression in the brain or in the testes at 12h or 24h post injection, we cannot discard the possibility for rapid and transient effects occurring between and 12h of treatment. Nevertheless, future studies will help to clarify a possible role of E2 in the regulation of GALRs in sea bass. In summary, orthologous gene duplicates of vertebrate GALR1 and GALR2 were isolated in sea bass and phylogenetic analysis revealed previously unreported GALR2 orthologs in teleost fish and tetrapods. All dlGALRs are expressed in male and female brain and the expression of dlGALR1a and dlGALR1b appears to be sexually dimorphic, with higher expression in testis than in ovary. Finally, the expression of both receptors was up regulated by 11KT in pre-pubertal sea bass testis. These results suggest that the galaninergic system, in particular the GALR1 isoforms may play a role in sea bass male reproductive function.