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

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.

The influence of the amino acid substitution I98K on the catalytic activity of baboon cytochrome P450 side-chain cleavage (CYP11A1).

Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the first and "rate-limiting" step in steroidogenesis, the conversion of cholesterol to pregnenolone. In an effort to gain further insight into the structure/function relationship of this key enzyme, CYP11A1 was characterized in the Cape baboon (Papio ursinus), a species closely related to humans. Baboon cDNA was isolated from adrenal tissue and direct sequence analysis showed mature baboon and human CYP11A1 share 98% deduced amino acid homology. The cDNA was subsequently amplified and two recombinant constructs, CYP11A1a and CYP11A1b, were cloned. Sequence analyses of the constructs revealed four amino acid substitutions. The constructs were expressed in nonsteroidogenic mammalian COS-1 cells with 25-hydroxycholesterol as substrate. Apparent Km values of 1.62 and 4.53 microM were determined for CYP11A1a and CYP11A1b, respectively. Homology modeling revealed that the lower substrate affinity of CYP11B1b could be attributed to an I98K substitution, which lies between the B and C helices, providing further evidence for the importance of this domain in the catalytic activity of CYP11A1.