Hepatic expression profiling in smolting and adult coho salmon (Onchorhynchus kisutch)

Abstract

Coho salmon are a critical Pacific salmon species that undergo complex physiological transformations as they migrate towards seawater and enter adult life stages. During these periods, coho may receive exposure to waterborne pollutants that coincide with outmigration through contaminated waterways and return to natal streams. However, little is known regarding the ontogenic modulation of gene expression during these critical life stages. Accordingly, the purpose of the present study was to characterize the hepatic transcriptome of smolting coho, adult males, and adult females by carrying out microarray analysis with a commercially available 16,000 cDNA element platform. Quantitative PCR (Q-PCR) analysis of genes involved in chemical biotransformation (cytochrome P450 isoforms 1A, and 2M1, glutathione S-transferase pi, microsomal GST), defense against metal exposure (metallothionein-A), and reproductive function (vitellogenin receptor) were developed for the purpose of analyzing specific genes of interest and to validate the microarray data. Microarray analysis identified 842 genes that were differentially expressed between smolts and adult males or females (p<0.001 and more than 2-fold difference). These 842 genes were not differentially expressed between adult males and females and, therefore, can be interpreted as a smolt-specific transcriptional profile. Of these 842 genes, 275 were well annotated and formed the basis for further bioinformatics analysis. Many of the differentially expressed genes were involved in basic cellular processes related to protein biosynthesis and degradation (24%), ion transport (12%), transcription (8%), cell structure (8%) and cellular energetics (6%). The majority of differentially expressed genes involved in signal transduction and energy metabolism were expressed at higher levels in adult coho relative to smolts. However, genes associated with cellular protection against chemical injury (i.e. biotransformation, DNA damage repair, and protection against oxidative stress) did not generally differ among the groups. Q-PCR studies revealed extensive interindividual variation in mRNA expression, but were consistent with the microarray results (R(2)=0.74). Collectively, our results indicate differences in liver gene expression in young smolting coho salmon relative to adults and extensive interindividual variation in biotransformation gene expression. However, we did not find a global lack of hepatic biotransformation capacity or poor cellular detoxification response capacity in smolting cohos based on mRNA profiles.

Fig 1

Venn diagram of the number of differentially expressed genes among adult males, adult females and smolting salmon using a cutoff of p<0.001 only. As observed, the diagram depicts 842 genes that were differentially expressed among males and females relative to smolts, 309 genes that were differentially expressed among females and smolts, 368 genes differentially expressed among males and smolts, and 75 genes that were differentially expressed among males and females.

Fig 2

Pie chart with biological function of annotated genes that were differentially expressed among smolting salmon in adults at p<0.001 and using a 2-fold cutoff value.

Figure 3

Q-PCR analysis of CYP1A and 2M1 mRNA gene expression in coho salmon of different life stages. Values represent the mean ±SD for n=4-6 animals for each group with all Q-PCR assays conducted in triplicate incubations. Values sharing different letter superscript are significantly different than their corresponding values atp≤ 0.05. Full gene names for gene labels are found in Table 2.

Figure 4

Q-PCR analysis of GST pi, mGST, metallothionein A, and vitellogenin receptor mRNA gene expression in coho salmon of different life stages. Values represent the mean±SD for n=4-6 animals for each group with all Q-PCR assays conducted in triplicate incubations. Values sharing different letter superscript are significantly different than their corresponding values at p≤ 0.05. Full gene names for gene labels are found in Table 1.

Figure 5

Correlation among gene expression data obtained by microarray analysis and Q-PCR. Values represent the mean mRNA expression values for CYP1A, CYP2M1, mGST, GST pi and VTG-receptor normalized to the expression of β-globin for all individuals and groups, with the Q-PCR data on the X-axis and the microarray data on the y-axis. A correlation value of R²=0.74 (p≤0.05) was obtained by linear regression analysis.