Substantial Downregulation of Myogenic Transcripts in Skeletal Muscle of Atlantic Cod during the Spawning Period

Abstract

Gonadal maturation is an extremely energy consuming process for batch spawners and it is associated with a significant decrease in growth and seasonal deterioration in flesh quality. Our knowledge about the molecular mechanisms linking sexual maturation and muscle growth is still limited. In the present study, we performed RNA-Seq using 454 GS-FLX pyrosequencing in fast skeletal muscle sampled from two-year-old Atlantic cod (Gadus morhua) at representative time points throughout the reproductive cycle (August, March and May). In total, 126,937 good quality reads were obtained, with 546 nucleotide length and 52% GC content on average. RNA-Seq analysis using the CLC Genomics Workbench with the Atlantic cod reference UniGene cDNA data revealed 59,581 (46.9%) uniquely annotated reads. Pairwise comparison for expression levels identified 153 differentially expressed UniGenes between time points. Notably, we found a significant suppression of myh13 and myofibrillar gene isoforms in fast skeletal muscle during the spawning season. This study uncovered a large number of differentially expressed genes that may be influenced by gonadal maturation, thus representing a significant contribution to our limited understanding of the molecular mechanisms regulating muscle wasting and regeneration in batch spawners during their reproductive cycle.

Fig 1. Growth history of Atlantic cod throughout their first reproductive cycle.

At the start of the experiment the fish were two years old (males, n = 49; females, n = 84). Specific growth rate SGR (%) is indicated by horizontal line (males) and dotted line (females). Somatic condition factor (K) is displayed by black dots (males) and white diamonds (females). The bar underneath indicate the light regime corresponding to the natural environmental photoperiod conditions in Bodø (67°N), Norway. Sea water temperature is represented by blue diamonds with a dashed line. Asterisks (*) indicates significant differences between sexes at a particular sampling point at P < 0.05 (two-tailed t-test). Different superscript letters highlight significant differences within the same sex throughout the reproductive cycle at P < 0.05 (Tukey's multiple comparison test; lower-case letters: males; capital letters: females). Sampling points of fast skeletal muscle for 454 pyrosequencing are indicated by yellow circles.

Fig 2

(a) Sampling positon of fast skeletal muscle and (b) schematic representation of the data processing pipeline for annotation, gene ontology, heat map, and differential expression analyses of Atlantic cod fast skeletal muscle transcriptomes for both sexes throughout the year.

Fig 3. Overview of functional annotation results of Atlantic cod fast skeletal muscle transcriptome.

Analysis was performed by Blast2GO-PRO software. a) Distribution of annotated sequence reads to UniGene cDNA reference data. b) Functional annotation results of Atlantic cod fast skeletal muscle transcriptome annotated to UniGene cDNA reference data.

Fig 4. Gene ontology (GO) analysis of Atlantic cod fast skeletal muscle transcriptome annotated to UniGene cDNA reference data.

Analysis was performed with Blast2GO-PRO. a) Distribution of transcripts with major category of molecular functions at level 2. b) Distribution of transcripts with major category of cellular component. c) Distribution of transcripts with major category of biological process. The larger pie charts display the combined transcript data from six libraries, whereas smaller pie charts indicate individual data for each library.

Fig 5. Heat map of the top 50 highly expressed genes.

The map contains 50 rows and 6 columns corresponding to the top 50 highly expressed genes and sampling points for both sexes, respectively. All 50 genes have individual UniGene accession numbers and their common gene name registered in NCBI. Several UniGenes (e.g., myosin heavy chain and titin) share the same common gene name but are referred to as individual isoforms, as long as they have unique UniGene accession numbers. Typical clusters containing genes that show similar expression patterns are separated by a dashed line and their expression patterns throughout the year are summarised with arrow graphs for both females (pink arrows) and males (blue arrows).

Fig 6. Differentially expressed genes (DEGs) in fast skeletal muscle of female Atlantic cod between August and March.

DEGs were determined by pairwise comparison when their log₂ fold change was log₂ Ratio > 2 (upregulated gene) or log₂ Ratio < -2 (downregulated gene). X- and y-axis indicate the log₂ (RPKM values of female muscle in August) and log₂ (RPKM values of female muscle in March), respectively. Green circles represent upregulated DEGs and red circles indicate downregulated DEGs. Grey circles show the UniGenes that did not display significant changes.

Fig 7. Phylogenetic inference of myosin heavy chain (myh) genes in vertebrates.

Red and blue asterisks indicated the myh clones annotated to UniGene and Ensembl reference data sets, respectively. Numbers at the nodes indicate posterior probability values obtained from the Bayesian analysis. Ensembl transcript IDs and NCBI accession nos. of teleost myh13 genes can be found in S6 and S7 Tables, respectively.

Fig 8. qPCR quantification of five DEGs in fast skeletal muscle throughout a reproductive cycle and comparison with RPKM values from RNA-Seq.

Pink and blue bars show the relative mRNA expression in females and males, respectively. Green dots indicate RPKM values from the RNA-Seq. Error bars show S.E.M. at each point (n = 6 for each sex). Different superscript letters indicate significant differences within sex at p < 0.05.