Blubber transcriptome responses to repeated ACTH administration in a marine mammal

Abstract

Chronic physiological stress impacts animal fitness by catabolizing metabolic stores and suppressing reproduction. This can be especially deleterious for capital breeding carnivores such as marine mammals, with potential for ecosystem-wide effects. However, the impacts and indicators of chronic stress in animals are currently poorly understood. To identify downstream mediators of repeated stress responses in marine mammals, we administered adrenocorticotropic hormone (ACTH) once daily for four days to free-ranging juvenile northern elephant seals ( Mirounga angustirostris ) to stimulate endogenous corticosteroid release, and compared blubber tissue transcriptome responses to the first and fourth ACTH administrations. Gene expression profiles were distinct between blubber responses to single and repeated ACTH administration, despite similarities in circulating cortisol profiles. We identified 61 and 12 genes that were differentially expressed (DEGs) in response to the first ACTH and fourth administrations, respectively, 24 DEGs between the first and fourth pre-ACTH samples, and 12 DEGs between ACTH response samples from the first and fourth days. Annotated DEGs were associated with functions in redox and lipid homeostasis, suggesting potential negative impacts of repeated stress on capital breeding, diving mammals. DEGs identified in this study are potential markers of repeated stress in marine mammals, which may not be detectable by endocrine profiles alone.

A blubber gene expression index for evaluating stress in marine mammals

“…Baseline sample collection was conducted as previously described ( Deyarmin et al , 2019 , McCormley et al , 2018 ) during September to November of 2017 at Año Nuevo State Reserve, San Mateo County, CA, USA. This period is generally a short haul out and is not associated with extensive fasting, molting or reproduction.…”

“…The inner half of each blubber sample (closest to muscle) was used for RNA extraction as this tissue layer is considered more metabolically active than outer blubber in marine mammals ( Ball et al , 2017 , Struntz et al , 2004 ). In addition, inner blubber was used for elephant seal transcriptomes that identified the markers used in this study ( Deyarmin et al , 2019 , Khudyakov et al , 2017 ). Blubber tissue (~100 mg) was minced with a sterile scalpel on dry ice and homogenized in 1 mL of Qiazol (Qiagen, USA) by bead beating in a Bullet Blender Storm 24 instrument (Next Advance, USA) for two 2-min cycles.…”

“…Primers for qPCR assays were designed to target genes that were previously identified as differentially expressed in elephant seal blubber in response to repeated ACTH administration ( Deyarmin et al , 2019 ). Candidate genes were selected based on three characteristics: (i) significant BlastX hits to a protein with known function in the UniProt SwissProt database, (ii) high transcript abundance (number of transcripts per million, TPM ≥ 20) and (iii) relatively consistent expression levels between biological replicates (mean CV for replicate TPM = 24.4%).…”

“…As marine mammal blubber is vertically stratified by fatty acid composition, cellular morphology, rate of lipid accumulation and mobilization and gene expression ( Ball et al , 2017 , Guerrero et al , 2017 , Struntz et al , 2004 ), the impacts of stress hormones are likely to be more pronounced in the inner, more metabolically active layer of blubber. We previously identified a cluster of genes that were differentially expressed in inner blubber in response to acute and repeated HPA axis stimulation in a model marine mammal, the northern elephant seal ( Mirounga angustirostris ; Deyarmin et al , 2019 , McCormley et al, 2018 , Khudyakov et al , 2017 ). These included upregulated genes encoding metabolic and antioxidant enzymes (long-chain-fatty-acid CoA ligase 1, ACSL1 ; hydroxymethylglutaryl-CoA synthase 2, HMGCS2 ; cysteine dioxygenase 1, CDO1 ; E3-SUMO-protein ligase PIAS4, PIAS4 ; glutathione peroxidase 3, GPX3 ; microsomal glutathione S-transferase 1, MGST1 ), adipokines (adiponectin, ADIPOQ ; leptin, LEP ), pro-adipogenic factors (dickkopf-related protein 1, DKK1 ; zinc-alpha-2-glycoprotein 1, AZGP1 ), lipid droplet proteins (perilipin 1, PLIN1 ; cell death activator CIDE-A, CIDEA ) and other metabolism-regulating proteins (glycine receptor subunit alpha-2, GLRA2 ), and a gene associated with extracellular matrix remodeling (transforming growth factor beta-induced, TGFBI ), which was downregulated ( Deyarmin et al ., 2019 ).…”

“…We previously identified a cluster of genes that were differentially expressed in inner blubber in response to acute and repeated HPA axis stimulation in a model marine mammal, the northern elephant seal ( Mirounga angustirostris ; Deyarmin et al , 2019 , McCormley et al, 2018 , Khudyakov et al , 2017 ). These included upregulated genes encoding metabolic and antioxidant enzymes (long-chain-fatty-acid CoA ligase 1, ACSL1 ; hydroxymethylglutaryl-CoA synthase 2, HMGCS2 ; cysteine dioxygenase 1, CDO1 ; E3-SUMO-protein ligase PIAS4, PIAS4 ; glutathione peroxidase 3, GPX3 ; microsomal glutathione S-transferase 1, MGST1 ), adipokines (adiponectin, ADIPOQ ; leptin, LEP ), pro-adipogenic factors (dickkopf-related protein 1, DKK1 ; zinc-alpha-2-glycoprotein 1, AZGP1 ), lipid droplet proteins (perilipin 1, PLIN1 ; cell death activator CIDE-A, CIDEA ) and other metabolism-regulating proteins (glycine receptor subunit alpha-2, GLRA2 ), and a gene associated with extracellular matrix remodeling (transforming growth factor beta-induced, TGFBI ), which was downregulated ( Deyarmin et al ., 2019 ). Gene expression changes were correlated with other markers, including increase in cortisol, aldosterone and reverse triiodothyronine (rT3) levels and decrease in total triiodothyronine (tT3) levels ( McCormley et al , 2018 ).…”