The stress steroid system of the Pacific hagfish, Eptatretus stoutii
Creators: Frost, Christiana R.
Publisher: name: University of Alberta Library
Year: 2022

<p>The Pacific hagfish, Eptatretus stoutii, is a basal, jawless marine fish considered key to understanding the evolution of vertebrates as is their sister group, the lampreys.  Cyclostomes like hagfishes and lampreys differ from gnathostomes in their stress response system including the hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis.  For hagfishes, little is known about the identity of their primary corticosteroid(s), steroidogenic enzyme(s), and steroidogenic tissue(s).<br>
Through bioinformatic analyses, including ancestral character reconstruction (ACR), cytochrome p450 (CYPs) and hydroxysteroid dehydrogenase (HSDs) enzymes involved in the stress steroid synthesis pathway were examined as traits in fishes to predict the primary corticosteroid pathway of hagfishes.  Hagfishes were predicted to possess genes for 3𝛽-HSD, CYP11a1, CYP17, and CYP21.  The presence of these implies that hagfishes could synthesize 11-deoxycorticosterone (DOC) and 11-deoxycortisol (11-DOC).  Previous studies, however, did not detect DOC nor 11-DOC in hagfish plasma.<br>
Physiological experiments were also performed on Pacific hagfish.  These experiments involved injecting hagfish with steroid implants containing an upstream steroid (pregnenolone or progesterone) early in the steroid synthesis pathway to induce mineralocorticoid and glucocorticoid responses.  Fish were also exposed to either an acid (HCl) injection or an exercise stressor (30 minutes of handling) to induce a stress response.  Hagfish plasma analyzed via targeted liquid chromatography tandem mass spectroscopy (LC-MS/MS) detected pregnenolone and progesterone in the low concentration range (&lt; 3 ng/mL) in stressed fish without steroid injection.  The detection of these two steroid products present in the steroidogenesis pathway implies the presence of two key steroidogenic enzymes, CYP11a1 and 3𝛽-HSD, in hagfish.  No cortisol was detected by LC-MS/MS thereby providing further support that cortisol is not a corticosteroid found in hagfish and that CYP11b, the enzyme that assists in the synthesis of cortisol, is absent or not expressed by hagfish.  It should be noted that steroid standards for DOC and 11-DOC were not present in this analysis, and therefore, we could not determine whether these steroids were present or absent from Pacific hagfish.<br>
Preliminary work to perform real-time quantitative polymerase chain reaction (RT-qPCR) resulted in four reference genes suitable for mapping gene expression in the gonad, the gut (fore-, mid-, and hindgut regions), and the kidney (upper and lower regions).  These primers can be used to continue the investigation of the expression of steroidogenic enzymes of hagfish tissues.<br>
In response to the exercise stressor, plasma glucose concentrations in hagfish increased.  Significant increases in plasma glucose levels in exercised fish without steroid injection ranged from 0.8 – 9.5 mM on average over the time course of the experiment (48 h).  While exercised pregnenolone and progesterone-injected fish had plasma glucose levels ranging from 0.4 – 5.5 mM and 0.6 – 6.4 mM over 48 h, respectively.  The injection of either pregnenolone or progesterone resulted in a decline of the gluconeogenic response in hagfish compared to exercise-stressed fish without any steroid injection, suggesting that these steroids have a suppressive effect on glucose release.<br>
    In response to the acid stressor, all fish experienced metabolic acidosis (mean pH 6.4 – 6.8, bicarbonate ion concentration ranging 2.12 – 5 mmol/L) within the first 2 h post treatment.  Morphometric analysis of hagfish gills at 48 h post-acid stressor demonstrated a decrease in the thickness of the lamellar epithelium compared to control fish.  Changes in lamellar microplicae due to the acid stressor were also observable via scanning electron microscopy.  Fish treated with the pregnenolone injection and acid had elongated microplicae compared to control fish microplicae that were shorter and rounder in appearance.<br>
The findings of this research demonstrate the advancement of methods and understanding of the stress steroid system of the Pacific hagfish.  Hagfish do not possess cortisol as their predominant corticosteroid thereby demonstrating a novel stress steroid profile that is distinct from the rest of the gnathostome lineage.</p>