Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 Down-regulation of both isotocin (confirmed by qPCR) and vasotocin was evident in the brain of precocious male fish, however all three clones representing these genes on the array were also differentially expressed between the two NPB pools (P < 0.01). This suggests some important variation in the ten non-precocious individuals that made up the two pools. Up-regulation of vasotocin, and to a lesser extent isotocin, is more often associated with reproduction especially through the known effects of vasotocin on social behaviour [52,53]. However, differences in the effects of vasotocin administration on aggression and/or courtship are clearly seen between fish species with some showing increased and others decreased aggression [54-56]. There are also seasonal variations in transcript levels of both vasotocin and isotocin in, for example, the masu salmon which in addition show changes between imma- ture parr and precocious males [57]. It has been observed that the protein levels of these hormones do not correspond to the levels of their mRNAs - vasotocin transcript levels are low in Nov in immature parr when vasotocin protein levels are near maximal [57]. Further- more, vasotocin is also involved in osmoregulation, car- diovascular activity, stress and metabolism [58]. Because precocity possibly interferes with normal progress through the parr-smolt transformation, the relative decrease in vasotocin we observe in precocious fish may be linked to an early regulatory effect on osmoregula- tion, and the variation we observe in the non-precocious individuals may be due to early preparation for smolting in some fish. Another gene that appeared to be up-regulated (1.2- fold) in the brain was melanin-concentrating hormone (MCH). MCH is expressed as a pro-hormone mainly in the hypothalamus and processed in the pituitary where the mature short peptide (17 amino acids in most tele- osts) stimulates the aggregation of melanosome in mela- nophores resulting in the silvering of the fish [59]. This is somewhat at odds with the expected inhibition of sil- vering in precocious fish where the parr-smolt transfor- mation is delayed. Receptors for MCH are, however, distributed much more widely than the brain and hypothalamus suggesting that the functional roles of MCH (which may include regulating food intake) are much more diverse than melanin concentration [59,60]. The DNA sequences of the MCH1 and MCH2 isoforms of Chinook salmon MCH are very similar and are unli- kely to be distinguished on the microarray. Because both MCH isoform sequences are not available for Salmo salar, two fragments were isolated and sequenced from Salmo salar cDNA using primers based on the Chinook sequences [61]. Quantitative PCR (using pri- mers designed to the two Atlantic salmon fragment sequences) of individual samples from the PB2 and Page 11 of 18 NPB2 pools showed approximate 1.7-fold up-regulation of MCH1 and 1.4-fold up-regulation of MCH2 in the precocious samples. MCH2 levels (Ct~34) were at least 100 times lower than MCH1 levels (Ct~26) so compari- sons of MCH2 levels between individuals were not reli- able; however there was still considerable variability in the levels of MCH1 in the ten individuals. An approxi- mate 290-fold difference (Additional file 2) between the highest and the lowest individuals may reflect sampling inconsistency where some specialised regions of the brain may have been mistakenly included (although the pituitary and hypothalamus had been purposefully dis- sected from all brain samples). Alpha- and beta-globin gene expression was down-regu- lated in the precocious brain as they had been in the testes. Surprisingly all eight globin clones were also differ- entially expressed between the two pools of precocious brains (PB1 and PB2); this was eight of the nine sequenced clones in a list of twelve clones (P < 0.01). Although the melting point curve on qPCR showed a single peak, it was noticeable during the optimisation of beta-globin primers that there were at least two forms amplified. Haemoglobin multiplicity in teleosts is common: embryonic forms give way to adult forms and it also has been suggested that fish which travel great distances across temperature differen- tials show more multiplicity [62]. There are some interesting similarities and differences between our precocious males and those of Aubin- Horth [39,41]. Both studies show down-regulation of genes in precocious males was more pronounced than up-regulation; we see down-regulation of glutamine synthetase and RpL13A which ties in with the above study but we also see down-regulation of vasotocin, iso- tocin and type II keratin K8 which seems at odds with the Aubin-Horth study. Also alpha- and beta-globin are oppositely regulated in both studies. Possibly the differ- ences seen in the two studies relates to the different times of fish sampling which were October for the Aubin-Horth study and three months later in January for our study. We chose to collect both groups of males in January when precocity was externally evident (size, body shape, milt production). In January the testes could therefore be best described as mature, possibly on the point of regression whereas in October they are likely to be recrudescent. Since maturation is controlled by the HPG axis, in October the brain will be signalling the gonads to mature and to begin steroidogenesis, and accompanying this there will ensue a range of beha- vioural changes. Therefore it is possibly not surprising that the Aubin-Horth study shows up-regulation of genes involved in reproduction and this study shows some opposite effects. Furthermore, our control group (immature males) may be preparing for the parr-smolt transformation