Cortisol is the main glucocorticoid hormone promoting compensatory metabolic responses of stress in teleosts. This hormone acts through genomic and membrane-initiated actions to exert its functions inside the cell. Experimental approaches, using exogenous cortisol administration, confirm the role of this hormone during short (minutes to hours)- and long-term (days to weeks) responses to stress. The role of membrane-initiated cortisol signaling during long-term responses has been recently explored. In this study, Sparus aurata were intraperitoneally injected with coconut oil alone or coconut oil containing cortisol, cortisol-BSA, or BSA. After 3 days of treatment, plasma, liver, and skeletal muscle were extracted. Plasma cortisol, as well as metabolic indicators in the plasma and tissues collected, and metabolism-related gene expression, were measured. Our results showed that artificially increased plasma cortisol levels in S. aurata enhanced plasma glucose and triacylglycerols values as well as hepatic substrate energy mobilization. Additionally, cortisol stimulated hepatic carbohydrates metabolism, as seen by the increased expression of metabolism-related genes. All of these responses, observed in cortisol-administered fish, were not detected by replicating the same protocol and instead using cortisol-BSA, which exclusively induces membrane-initiated effects. Therefore, we suggest that after three days of cortisol administration, only genomic actions are involved in the metabolic responses in S. aurata.

The production and release of cortisol during stress responses are key regulators of growth in teleosts. Understanding the molecular responses to cortisol is crucial for the sustainable farming of rainbow trout (Oncorhynchus mykiss) and other salmonid species. While several studies have explored the genomic and non-genomic impacts of cortisol on fish growth and skeletal muscle development, the long-term effects driven by epigenetic mechanisms, such as cortisol-induced DNA methylation, remain unexplored. In this study, we analyzed the transcriptome and genome-wide DNA methylation in the skeletal muscle of rainbow trout seven days after cortisol administration. We identified 550 differentially expressed genes (DEGs) by RNA-seq and 9059 differentially methylated genes (DMGs) via whole-genome bisulfite sequencing (WGBS) analysis. KEGG enrichment analysis showed that cortisol modulates the differential expression of genes associated with nucleotide metabolism, ECM-receptor interaction, and the regulation of actin cytoskeleton pathways. Similarly, cortisol induced the differential methylation of genes associated with focal adhesion, adrenergic signaling in cardiomyocytes, and Wnt signaling. Through integrative analyses, we determined that 126 genes showed a negative correlation between up-regulated expression and down-regulated methylation. KEGG enrichment analysis of these genes indicated participation in ECM-receptor interaction, regulation of actin cytoskeleton, and focal adhesion. Using RT-qPCR, we confirmed the differential expression of lamb3, itga6, limk2, itgb4, capn2, and thbs1. This study revealed for the first time the molecular responses of skeletal muscle to cortisol at the transcriptomic and whole-genome DNA methylation levels in rainbow trout.

In aquaculture, many stressors can negatively affect growth in teleosts. It is believed that cortisol performs glucocorticoid and mineralocorticoid functions because teleosts do not synthesize aldosterone. However, recent data suggest that 11-deoxycorticosterone (DOC) released during stress events may be relevant to modulate the compensatory response. To understand how DOC modifies the skeletal muscle molecular response, we carried out a transcriptomic analysis. Rainbow trout (Oncorhynchus mykiss) were intraperitoneally treated with physiological doses of DOC in individuals pretreated with mifepristone (glucocorticoid receptor antagonist) or eplerenone (mineralocorticoid receptor antagonist). RNA was extracted from the skeletal muscles, and cDNA libraries were constructed from vehicle, DOC, mifepristone, mifepristone plus DOC, eplerenone, and eplerenone plus DOC groups. The RNA-seq analysis revealed 131 differentially expressed transcripts (DETs) induced by DOC with respect to the vehicle group, mainly associated with muscle contraction, sarcomere organization, and cell adhesion. In addition, a DOC versus mifepristone plus DOC analysis revealed 122 DETs related to muscle contraction, sarcomere organization, and skeletal muscle cell differentiation. In a DOC versus eplerenone plus DOC analysis, 133 DETs were associated with autophagosome assembly, circadian regulation of gene expression, and regulation of transcription from RNA pol II promoter. These analyses indicate that DOC has a relevant function in the stress response of skeletal muscles, whose action is differentially modulated by GR and MR and is complementary to cortisol.

Cortisol, the principal glucocorticoid in teleosts, coordinates the physiological response to stress by regulating metabolism, protein turnover, and growth. Although the molecular effects of acute cortisol exposure have been extensively characterized, the epigenomic mechanisms underlying skeletal muscle remodeling during prolonged cortisol elevation remain poorly understood. Here, we investigated how chronic cortisol reshapes chromatin accessibility and gene expression in rainbow trout (Oncorhynchus mykiss) skeletal muscle using an integrative ATAC-seq and RNA-seq approach. Fish received a single intraperitoneal injection of cortisol (50 μg g⁻¹ body weight) formulated in coconut oil for sustained hormone release and were sampled after 14 days. Cortisol treatment significantly increased plasma cortisol levels, reduced specific growth rate, and increased skeletal muscle protein carbonylation, indicating impaired growth and oxidative protein damage. ATAC-seq revealed extensive chromatin remodeling, including 126,780 cortisol-specific accessible regions and 3393 genes associated with increased accessibility. These genes were enriched in pathways related to FoxO signaling, autophagy, and apoptosis, whereas regions with reduced accessibility were associated with TGF-β signaling, extracellular matrix interactions, and muscle structural maintenance. RNA-seq identified 2170 differentially expressed genes (844 upregulated and 1326 downregulated), revealing a parallel transcriptional shift toward protein turnover, autophagy, and metabolic adaptation, together with repression of extracellular matrix organization, focal adhesion, integrin signaling, and cytoskeletal organization. Integrative analysis identified 113 genes with coordinated increases and 46 genes with coordinated decreases in chromatin accessibility and transcript abundance, highlighting ubiquitin-mediated proteolysis, mitophagy, FoxO signaling, and metabolic pathways as core components of the chronic cortisol response. Collectively, these findings demonstrate that sustained cortisol elevation drives coordinated epigenomic and transcriptomic remodeling of skeletal muscle, promoting a catabolic phenotype characterized by enhanced protein turnover, oxidative protein damage, and impaired structural maintenance, ultimately contributing to reduced growth and potentially compromising fish welfare under aquaculture conditions.