Dysregulation of testis mRNA expression levels in hatchery-produced vs wild greater amberjack Seriola dumerili

Abstract

Reproductive dysfunctions have been recently documented in male greater amberjack Seriola dumerili caught from the wild and reared in captivity. In the present study, we compared testis transcriptome in wild fish (WILD), hatchery-produced fish with apparently normal spermatogenesis (Normal Farmed; NormalF) and hatchery-produced fish with evident reproductive dysfunction (Dysfunctional Farmed; DysF). Gene expression analysis identified 2157, 1985 and 74 differentially expressed genes (DEGs) in DysF vs WILD, NormalF vs DysF and NormalF vs WILD comparisons, respectively. In DysF, a dysregulation of several interconnected biological processes, including cell assembly, steroidogenesis and apoptosis was found. Gene enrichment of progesterone-mediated oocyte maturation, oocyte meiosis and cell cycle pathways were identified in the DysF vs NormalF comparison. Most of the DEGs involved in the enriched pathways were downregulated in DysF. The comparison of NormalF vs WILD showed that most of the DEGs were downregulated in NormalF, including a gene that encodes for a regulatory protein with a protective role in apoptosis regulation (ptpn6), indicating that spermatogenesis was dysfunctional also in the apparently "normal" hatchery-produced fish. Hence, rearing of male greater amberjack in captivity, from eggs produced by captive breeders, did not prevent the appearance of reproductive dysfunctions, and these dysfunctions involved several biological processes and metabolic pathways.

Figure 1

Schematic representation of the experimental design. (1) Testis samples were taken from wild and hatchery-produced greater amberjack. (2) The reproductive state was assessed through the histological analysis of the testes. (3) Fish were then divided in three groups based on their origin and reproductive state: WILD (wild fish showing normal spermatogenesis), NormalF (hatchery-produced fish showing normal spermatogenesis); DysF (hatchery-produced fish showing altered spermatogenesis). (4) Testis RNA was extracted and sequenced. (5) Differentially expressed genes (DEGs) between groups were identified. (6) Functional analysis of DEGs was carried out. * After the evaluation of the RNA quality, one of the wild samples was excluded from further analyses.

Figure 2

Micrographs of histological section from greater amberjack testes. (a, b) Wild specimen in active spermatogenesis phase (WILD group) showing large seminiferous tubules rich in spermatocysts. (c, d) Farmed specimen in active spermatogenesis with histological appearance similar to wild fish (NormalF group). (e, f) Farmed specimens showing arrested spermatogenesis (DysF group). Small seminiferous tubules with residual spermatocysts and luminal spermatozoa can be observed. Arrowheads indicate luminal spermatozoa; asterisks indicate spermatocysts. Hematoxylin–eosin staining. Bars = 100 μm in (b) and (d), 200 μm in (f), 300 μm in (a) and (c), 500 μm in (e).

Figure 3

Gonadosomatic index (a) and seminiferous tubule diameter (b) of wild greater amberjack (WILD), non-dysfunctional farmed fish (NormalF) and dysfunctional farmed fish (DysF). Different letters indicate statistically significant differences (Student’s t-test; P < 0.05).

Figure 4

VENN diagram of shared and unique genes related to testes samples of wild (WILD) and hatchery-produced greater amberjack, dysfunctional (DysF) and non-dysfunctional (NormalF).

Figure 5

VENN diagram of DEGs shared and unique among DysF vs NormalF, DysF vs WILD and NormalF vs WILD comparisons. DysF, dysfunctional farmed; NormalF, non-dysfunctional Farmed; WILD, wild greater amberjack.

Figure 6

Relationship between enriched pathways in testis samples from the three analysed groups. Pathways (nodes) are connected if they share 20% (default) or more genes. Darker nodes are more significantly enriched gene sets. Bigger nodes represent larger gene sets. Thicker edges represent more overlapped genes.

Figure 7

Protein–Protein interaction (PPi) network in DysF vs NormalF. The network was built using a confidence protein interaction (score = 0.7). Node background indicates gene upregulation (red, log2FC > 1.5) or downregulation (blue, log2FC < 1.5). Node contour indicates biological categories: male gamete generation (blue), gamete generation (yellow) spermatogenesis (green); KEGG: progesterone-mediated oocyte maturation (red), oocyte meiosis (pink), cell cycle (purple) pathways. Circles 1–4 are arbitrary representations of the main protein-protein interaction groups.

Figure 8

Protein–Protein interaction (PPi) network in DysF vs WILD. The network was built using a confidence protein interaction (score = 0.7). Node background indicates gene upregulation (red, log2FC > 1.5) or downregulation (blue, log2FC < 1.5). Node contour indicates biological categories: male gamete generation (blue), gamete generation (yellow) spermatogenesis (green), KEGG: Progesterone-mediated oocyte maturation (red), KEGG: Oocyte meiosis (pink) KEGG: Cell cycle (purple). Circles 1–4 are arbitrary representations of the main protein-protein interaction groups.

Figure 9

Protein–Protein interaction (PPi) networks in NormalF vs WILD. Networks were built using a medium confidence protein interaction (score = 0.4). Nodes are coloured according to the following biological categories: regulation of tumor necrosis factor production (red); cell killing (pink); interspecies interaction between organisms (gray); sequestering of calcium ion (green); immune system process (orange).