11-Deoxycorticosterone (DOC)'s Action on the Gill Osmoregulation of Juvenile Rainbow Trout (Oncorhynchus mykiss)

Abstract

In aquaculture, stress can negatively affect fish growth. For years, the cortisol hormone has been thought to play both glucocorticoid and mineralocorticoid functions. Nevertheless, recent research has suggested that 11-deoxycorticosterone (DOC) released during stress could contribute to cortisol actions, though this process is still misunderstood. Here, we evaluated the DOC effects on physiological and early transcriptional responses by RNA-seq. Juvenile rainbow trout were treated with DOC and/or glucocorticoids (mifepristone) or mineralocorticoid (eplerenone) receptor antagonists. Subsequently, plasma was collected, and cDNA libraries were generated from the gills of vehicle (control), DOC, mifepristone, mifepristone with DOC, eplerenone, and eplerenone with DOC groups. Calcium and phosphate levels in plasma were changed. Results revealed 914 differentially expressed transcripts (DETs) induced by DOC compared with control, mainly associated with sodium ion transmembrane transport, gluconeogenesis, negative regulation of transmembrane transport, and activation of innate immune response. DOC versus eplerenone with DOC comparison displayed 444 DETs related to cell-cell junction organization, canonical glycolysis, positive regulation of immune response, and potassium ion transport. Conversely, no DETs were detected in DOC versus mifepristone with DOC comparison. These data suggest that DOC has a relevant role in gill stress response and ion transport, which is differentially regulated by mineralocorticoid receptors.

Keywords: DOC; RNA-seq; gills; mineralocorticoid receptor; salmonid; stress response.

Figure 1

The plasma phosphate, calcium, chloride, and muscle water content of the rainbow trout. The phosphate (A), calcium (B), and chloride (C) plasma levels, as well as the (D) muscle water content, were measured in the fish treated with the vehicle, DOC, mifepristone, mifepristone + DOC, eplerenone, and eplerenone + DOC. The results are expressed as the means ± standard error of the means (SEM) (n = 5). Different letters represent the significant differences (p < 0.05) present when groups were compared.

Figure 1

The plasma phosphate, calcium, chloride, and muscle water content of the rainbow trout. The phosphate (A), calcium (B), and chloride (C) plasma levels, as well as the (D) muscle water content, were measured in the fish treated with the vehicle, DOC, mifepristone, mifepristone + DOC, eplerenone, and eplerenone + DOC. The results are expressed as the means ± standard error of the means (SEM) (n = 5). Different letters represent the significant differences (p < 0.05) present when groups were compared.

Figure 2

Venn diagram analysis. The Venn diagram indicates the numbers of differentially expressed transcripts under the vehicle vs. DOC and the DOC vs. eplerenone + DOC treatments (padj < 0.05 and absolute Log2FC > 1).

Figure 3

The gene ontology biological process (BP), molecular function (MF), and cellular component (CC) enrichment of the DETs in the vehicle and DOC treatments. (A) Upregulated DETs. (B) Downregulated DETs. The graph indicates the −log₁₀(p-value) that was enriched of the differentially expressed transcripts between the vehicle and DOC groups with a padj of <0.05.

Figure 4

The gene ontology biological process (BP), molecular function (MF), and cellular component (CC) enrichment of the differentially expressed transcripts between the DOC vs. eplerenone + DOC treatments. (A) Upregulated DETs. (B) Downregulated DETs. The graph indicates the −log₁₀(p-value) that was enriched by the differentially expressed transcripts between the group DOC vs. eplerenone + DOC with a padj of <0.05.

Figure 5

RNA-seq analysis validation via a real-time PCR of the DETs. The following DETs were selected for the real time-PCR validation of the RNA-seq process: egr1, cebpb, wnt11, idh2, aldoa, zfand5, cldn1, cldn6, ocln, cftr, slc24a5, kcnk1, nppa, cyp3a4, and oaz1. (A) Validation results between the vehicle vs. DOC and (B) the DOC vs. eplerenone + DOC. For the RNA-seq, “#” in blue indicates a padj of <0.05 and an absolute Log2FC of >1. For the real-time PCR (n = 3), the relative expression was normalized against actβ and fau, and the “*” in green indicates the significant differences in the fold change in the vehicle or DOC groups (mean ± SEM, n = 3, p < 0.05). The genes had the following abbreviations: egr1 (early growth response 1); cebpb (CCAAT enhancer binding protein beta); wnt11 (Wnt family member 11); idh2 (isocitrate dehydrogenase (NADP(+)) 2); aldoa (aldolase, fructose-bisphosphate A); zfand5 (zinc finger AN1-type containing 5); cldn1 (claudin 1); cldn6 (claudin 6); ocln (occludin); cftr (cystic fibrosis transmembrane conductance regulator); slc24a5 (solute carrier family 24 member 5); kcnk1 (potassium two-pore domain channel subfamily K member 1); nppa (natriuretic peptide A); cyp3a4 (cytochrome P450 family 3 subfamily A member 4); oaz1 (ornithine decarboxylase antizyme 1); fau (40S ribosomal protein S30); and actβ (beta-actin).