The initiation of puberty in Atlantic salmon brings about large changes in testicular gene expression that are modulated by the energy status

Abstract

Background: When puberty starts before males reach harvest size, animal welfare and sustainability issues occur in Atlantic salmon (Salmo salar) aquaculture. Hallmarks of male puberty are an increased proliferation activity in the testis and elevated androgen production. Examining transcriptional changes in salmon testis during the transition from immature to maturing testes may help understanding the regulation of puberty, potentially leading to procedures to modulate its start. Since differences in body weight influence, via unknown mechanisms, the chances for entering puberty, we used two feed rations to create body weight differences.

Results: Maturing testes were characterized by an elevated proliferation activity of Sertoli cells and of single undifferentiated spermatogonia. Pituitary gene expression data suggest increased Gnrh receptor and gonadotropin gene expression, potentially responsible for the elevated circulating androgen levels in maturing fish. Transcriptional changes in maturing testes included a broad variety of signaling systems (e.g. Tgfβ, Wnt, insulin/Igf, nuclear receptors), but also, activation of metabolic pathways such as anaerobic metabolism and protection against ROS. Feed restriction lowered the incidence of puberty. In males maturing despite feed restriction, plasma androgen levels were higher than in maturing fish receiving the full ration. A group of 449 genes that were up-regulated in maturing fully fed fish, was up-regulated more prominently in testis from fish maturing under caloric restriction. Moreover, 421 genes were specifically up-regulated in testes from fish maturing under caloric restriction, including carbon metabolism genes, a pathway relevant for nucleotide biosynthesis and for placing epigenetic marks.

Conclusions: Undifferentiated spermatogonia and Sertoli cell populations increased at the beginning of puberty, which was associated with the up-regulation of metabolic pathways (e.g. anaerobic and ROS pathways) known from other stem cell systems. The higher androgen levels in males maturing under caloric restriction may be responsible for the stronger up-regulation of a common set of (449) maturation-associated genes, and the specific up-regulation of another set of (421) genes. The latter opened regulatory and/or metabolic options for initiating puberty despite feed restriction. As a means to reduce the incidence of male puberty in salmon, however, caloric restriction seems unsuitable.

Keywords: Androgens; Nutrition; Puberty; Spermatogenesis; Testis; Transcriptomics.

Fig. 1

Characterization of the experimental groups. Androgen plasma levels (11KT, ng/mL) (a), gonadosomatic indices (b) and testicular levels of selected transcripts (c-f) analyzed in immature (imm) and maturing (mat) salmon receiving a normal feed ration (NR) or a restricted feed ration (RR). Data are expressed as mean ± SEM (N = 5–16; two-way ANOVA followed by Bonferroni’s test, * P < 0.05 at the α level of 0.05). RU, relative units

Fig. 2

Transcriptomic response at puberty of Atlantic salmon testis tissue sampled from males fed a normal (NR) or restricted ration (RR; 43% of NR). a Total numbers of DEGs (N = 5–16, indicated by grey numbers; Student’s t-test, P < 0.01 at the α level of 0.05, FDR < 0.05; fold change > │1.5│) in each pairwise comparison. Numbers next to the brackets give the number of total DEGs. b Up- and down-regulated genes identified during male salmon maturation independently of the food regime (right panel), or exclusively in males exposed to the restricted ration (left panel). For each comparison, black and grey letters represent the test condition and its corresponding control, respectively. c Venn diagram representation of transcripts differentially expressed in all pairwise comparisons performed. For each comparison, black and grey letters represent the test condition and its corresponding control, respectively. d Stimulatory effect on testicular gene expression during puberty for a set of 449 shared genes (one-way ANOVA followed by Tukey’s test, *** P < 0.001 at the α level of 0.05), and modulation of the magnitude of this effect by the feed ration

Fig. 3

Functional enrichment of maturation-induced gene expression in the salmon testis. a The data set containing modulated genes at puberty (i.e. 1262) was mapped (after GO enrichment analysis) resulting in a network of functionally related gene sets (red nodes) that form enrichment groups. Nodes represent statistically significant GO terms (P < 0.005 at the α level of 0.05, FDR < 0.01, overlap < 0.5) and links (grey edges) indicate the number of overlapping genes (indicated by their thickness) between connected sets. Groups of closely related GO terms are encircled with dashed lines and labeled. Number of identified genes is shown in brackets. b KEGG pathways identified in the maturing salmon testis. Each pathway shown is represented by at least 5 DEGs (P < 0.01 at the α level of 0.05, FDR < 0.05) and has a ratio of regulated genes (up−/down-, or vice versa) higher than 5. DEGs are highlighted with red (up-) or green (down-regulated) background

Fig. 4

Protein-protein interaction map for candidates found in pubertal males exposed to the restricted feed ration. The interaction network was generated using STRING database v10.5 (default settings; medium confidence of 0.4). A total number of 27 candidate genes was considered for the analysis. Proteins are represented as nodes, and lines indicate associations based on known functional interactions in zebrafish (see right bottom corner). The network is significantly enriched in interactions (P < 1.0E-16 at the α level of 0.05, FDR < 0.05). Red, purple and green nodes indicate proteins involved in Carbon, Purine and Pyrimidine metabolism, respectively. Nodes encircled in red color represent additional Carbon metabolism-associated proteins known in mammals (but not yet in zebrafish)

Fig. 5

Validation of microarray results by qPCR analysis. a Data are expressed as fold change with respect to the control group (experimental condition appearing as subscripts). Statistical significance (N = 6–15; Student’s t-test, P < 0.05 at the α level of 0.05) is highlighted with red (up-) or green (down-regulated) background. RR, restricted ration; NR, normal ration. b Correlation of microarray and qPCR. Comparison of changes in gene expression (represented as log2) derived from qPCR and microarray hybridizations revealed a significant correlation (Spearman’s rank test, P = 0.003 at the α level of 0.05)