Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 Page 6 of 18 Table 1: Differentially expressed genes of the testis during precocious maturation. (Continued) FE963911 GO256594 FE963864 ↑ ↓ ↓ 1.4 6.0 5.2 Similar to SIX homeobox 6 Transferrin Zinc finger protein Zic1 1E-90 3E-152 5E-143 DW628904 BT045182 NM_001140488 1Clones on this list may already have been sequenced in other projects (e.g. STRESSGENES). Accession numbers are given where available. 2An upward arrow indicates up-regulation in precocious males 3BLAST accession numbers starting with TC indicate sequences in the TIGR database. identified as an up-regulated gene in the precocious testes. Possibly ApoE is required for transport of choles- terol into the Leydig cell mitochondria in order to make available cholesterol for steroidogenesis or for transport of lipid needed either as a energy source or for cell membrane biosynthesis in gametogenesis [26]. Anti- Mullerian hormone (AMH: also known as Mullerian Inhibiting Substance) was strongly down-regulated in the precocious male testes. AMH has growth factor activity with a TGFbeta-like C-terminal domain and is known to have roles in Mullerian duct regression, sex determination, sex differentiation and gonad mesoderm development [27, 28]. Its role in testicular differentiation in fish was suggested when a spermatogenesis-prevent- ing substance was isolated from eel [29] and further reports confirm significant regulation in other fish [30,31]. Another up-regulated gene was gonadotropin- releasing hormone receptor (GnRH-R). The expression of gonadotropin-releasing hormones (GnRH) and their receptors in the gonads of fish and many other species has been reported previously [32] and suggests para- crine/autocrine control on gonad development. Although in fish there are multiple forms of GnRH pep- tides and GnRH receptors there appears to be one form of GnRH and GnRH-R in the gonad which varies in expression considerably depending on reproduction sta- tus [33]. Increased GnRH-R1, as reported in this study, is associated with the later stages of spermatogenesis in rainbow trout [33]. Another gene more often associated with the pituitary that shows differential expression in the testes is growth hormone (GH). Down-regulation, as seen here, probably relates to the full maturity of these testes (January) and the higher reported levels of recep- tors for growth hormone in recrudescent testes [30,34]; if gene transcription is being switched off lower levels might be expected that in the immature parr where levels may be quite steady or rising in preparation for the parr-smolt transformation [35]. on the same pools used for microarray hybridisations. Several potential housekeeping genes were analysed for use as a reference gene in quantitative PCR. Our goal was to identify a gene that could be reliably used as a reference in both the precocious samples and the adult maturing samples. Most of the genes examined (beta- actin, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine-guanine phosphoribosyl transferase, cyclo- philin and ubiquitin) were significantly differentially regulated in at least one developmental stage, but changes in succinate dehydrogenase (SD) expression levels were not significant (P > 0.05) (Additional file 2). Relative expression was therefore calculated using the Delta Delta C(t) method [36] using succinate dehydro- genase (SD) as the reference gene. Preliminary quantita- tive PCR experiments were set up to analyse the expression patterns of the genes from the panel using biological pools of non-precocious parr testes and pre- cocious parr testes (Figure 3a). Nine genes were selected for qPCR based mainly on the number of times the gene was independently identified on the array and on an examination of both fold changes and levels of signif- icance. Of the genes selected only cathepsin B showed a discrepancy between microarray analysis and qPCR. It appears that the microarray SSH clone was similar to GenBank accession no. NM_001140522 (S. salar cathe- psin B) but that the qPCR primers were designed to EST EG934102. This EST is also described as ‘cathepsin B’ but is a different isoform - different enough that the (reverse) primer is specific for this form. It is open to speculation why one form may be down-regulated more than 2-fold (qPCR) while, as the microarray suggests, another form appears up-regulated more than 2-fold. The fold changes in differential regulation were fre- quently larger by qPCR than by microarray: AMH levels by microarray analysis were ~6.5-fold down-regulated in precocity yet qPCR showed the fold down-regulation to be as much as 59-fold. Quantitative PCR on pooled precocious testis In order to confirm the findings of the microarray ana- lysis on the testes tissue a number of genes were selected and further investigated using real-time quanti- tative PCR. These qPCR experiments were carried out Quantitative PCR on individual testis After validation using fish pools, qRT-PCR of individual fish was carried out on a number of the genes (Figure 3b). As stated previously AMH was known to have roles in Mullerian duct regression and sex differentiation so it was