Expression and activity profiling of the steroidogenic enzymes of glucocorticoid biosynthesis and the fdx1 co‐factors in zebrafish

Abstract

The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing ferredoxin co-factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase-PCR and whole-mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a translational model with respect to understanding the roles of the genes for glucocorticoid biosynthesis and fdx co-factors during embryonic development and stress, as well as in brain homeostasis and function.

Glucocorticoid deficiency causes transcriptional and post-transcriptional reprogramming of glutamine metabolism

“…Both the generation of probes using gene-specific oligos summarized in Table S3 and whole-mount in situ hybridizations were carried out as previously described [ 77 ]. cyp17a2 expression was used to determine the size of the interrenal gland using Image J software.…”

“…They allow for comprehensive analysis of in vivo processes and for high-throughput compound screenings to identify novel drug targets. Zebrafish possess two paralogues of the human FDX gene, fdx1 and fdx1b , with fdx1b being specifically expressed in steroidogenic tissues [ 33 , 77 ] and serving as the only relevant electron donor for mitochondrial steroidogenesis. We have recently established an fdx1b zebrafish mutant line ( fdx1b −/− ) and showed that disruption of fdx1b leads to an impaired stress response and severe global cortisol deficiency in larvae [ 33 ].…”

Steroid Transport, Local Synthesis, and Signaling within the Brain: Roles in Neurogenesis, Neuroprotection, and Sexual Behaviors

“…Expression and activity studies in the brain of adult zebrafish were documented for 3α-Hsd, 3β-Hsd, 17β-Hsd, Cyp17, AroB, and 5α-reductase (Diotel et al, 2011b ). The recent reanalysis of the RP-HPLC profiles previously reported by Diotel et al ( 2011a ) indicated that [ 3 H]-pregnenolone can also be converted into cortisol and tetrahydrodeoxycorticosterone (THDOC), further highlighting Cyp21A2 and Cyp11C1 activities within the brain of adult zebrafish (Weger et al, accepted ).…”

Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae

“…Previous work also suggested that HIF promotes POMC activity in the mouse hypothalamic region(Zhang et al, 2011). On the other hand, Cyp17a2 is an enzyme involved in steroid hormone biosynthesis at the level of interrenal gland, which is activated upon ACTH stimulation(Ramamoorthy and Cidlowski, 2016;Eachus et al, 2017;Weger et al, 2018).…”