Aliesha Griffin

Redefining the initiation and maintenance of zebrafish interrenal steroidogenesis by characterizing the key enzyme cyp11a2.

Zebrafish are emerging as a model to study steroid hormone action and associated disease. However, steroidogenesis in zebrafish is not well characterized. Mammalian P450 side-chain cleavage enzyme (CYP11A1) catalyzes the first step of steroidogenesis, the conversion of cholesterol to pregnenolone. Previous studies describe an essential role for zebrafish Cyp11a1 during early development. Cyp11a1 has been suggested to be the functional equivalent of mammalian CYP11A1 in the zebrafish interrenal gland (equivalent to the mammalian adrenal), gonad, and brain. However, reported cyp11a1 expression is inconsistent in zebrafish larvae, after active cortisol synthesis commences. Recently a duplicated cyp11a gene, cyp11a2, has been described, which shares an 85% identity with cyp11a1. We aimed to elucidate the specific role of the two cyp11a paralogs. cyp11a1 was expressed from 0 to 48 hours post-fertilization (hpf), whereas cyp11a2 expression started after the development of the interrenal primordium (32 hpf) and was the only paralog in larvae. cyp11a2 is expressed in adult steroidogenic tissues, such as the interrenal, gonads, and brain. In contrast, cyp11a1 was mainly restricted to the gonads. Antisense morpholino knockdown studies confirmed abnormal gastrulation in cyp11a1 morphants. cyp11a2 morphants showed impaired steroidogenesis and a phenotype indicative of metabolic abnormalities. The phenotype was rescued by pregnenolone replacement in cyp11a2 morphants. Thus, we conclude that cyp11a1 is required for early development, whereas cyp11a2 is essential for the initiation and maintenance of zebrafish interrenal steroidogenesis. Importantly, this study highlights the need for a comprehensive characterization of steroidogenesis in zebrafish prior to its implementation as a model organism in translational research of adrenal disease.

Ferredoxin 1b (Fdx1b) Is the Essential Mitochondrial Redox Partner for Cortisol Biosynthesis in Zebrafish

Mitochondrial cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin 1 (FDX1) for catalytic activity. Key steps in steroidogenesis require mitochondrial CYP enzymes and FDX1. Over 30 ferredoxin mutations have been explored in vitro; however, no spontaneously occurring mutations have been identified in humans leaving the impact of FDX1 on steroidogenesis in the whole organism largely unknown. Zebrafish are an important model to study human steroidogenesis, because they have similar steroid products and endocrine tissues. This study aimed to characterize the influence of ferredoxin on steroidogenic capacity in vivo by using zebrafish. Zebrafish have duplicate ferredoxin paralogs: fdx1 and fdx1b. Although fdx1 was observed throughout development and in most tissues, fdx1b was expressed after development of the zebrafish interrenal gland (counterpart to the mammalian adrenal gland). Additionally, fdx1b was restricted to adult steroidogenic tissues, such as the interrenal, gonads, and brain, suggesting that fdx1b was interacting with steroidogenic CYP enzymes. By using transcription activator-like effector nucleases, we generated fdx1b mutant zebrafish lines. Larvae with genetic disruption of fdx1b were morphologically inconspicuous. However, steroid hormone analysis by liquid chromatography tandem mass spectrometry revealed fdx1b mutants failed to synthesize glucocorticoids. Additionally, these mutants had an up-regulation of the hypothalamus-pituitary-interrenal axis and showed altered dark-light adaptation, suggesting impaired cortisol signaling. Antisense morpholino knockdown confirmed Fdx1b is required for de novo cortisol biosynthesis. In summary, by using zebrafish, we generated a ferredoxin knockout model system, which demonstrates for the first time the impact of mitochondrial redox regulation on glucocorticoid biosynthesis in vivo.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator–like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic–pituitary–interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Adrenal Steroid Metabolites Accumulating in Congenital Adrenal Hyperplasia Lead to Transactivation of the Glucocorticoid Receptor

Patients with congenital adrenal hyperplasia (CAH) are often clinically less severely affected by cortisol deficiency than anticipated from their enzymatic defect. We hypothesize that adrenal steroid hormone precursors that accumulate in untreated or poorly controlled CAH have glucocorticoid activity and partially compensate for cortisol deficiency. We studied the in vitro effects of 17-hydroxyprogesterone (17OHP), progesterone (P), 21-deoxycortisol (21DF), and androstenedione (Δ4) on the human glucocorticoid receptor (hGR). Competitive binding assays were performed in HeLa cells. Nuclear translocation of the hGR was studied by transfection of COS-7 cells with a GFP-tagged hGR and fluorescence microscopy. Transactivation assays were performed in COS-7 cells and in HEK 293 cells after cotransfection with hGR and luciferase reporter vectors using a dual luciferase assay. 17OHP, P, and 21DF are able to bind to the hGR with binding affinities of 24-43% compared with cortisol. Δ4 has a low binding affinity. Incubation with 21DF led to complete nuclear translocation of the hGR, whereas treatment with 17OHP or P resulted in partial nuclear translocation. 21DF transactivated the hGR with an EC50 approximately 6 times the EC50 of cortisol. 17OHP and P transactivated the hGR with EC50s of more than 100 times the EC50 of cortisol. No hGR transactivation was detected after incubation with Δ4. 21DF, 17OHP, and P are able to bind, translocate, and transactivate the hGR in vitro and thus may have glucocorticoid activity. 21DF might have a clinically relevant agonistic effect on the hGR and could potentially partially compensate the cortisol deficiency in CAH patients.

Identification of a Novel Large CYP17A1 Deletion by MLPA Analysis in a Family with Classic 17α-Hydroxylase Deficiency

Steroid 17α-hydroxylase deficiency (17OHD) is a rare form of congenital adrenal hyperplasia caused by mutations in the 17α-hydroxylase (CYP17A1) gene. CYP17A1 is a key enzyme in the biosynthesis of adrenal and gonadal steroid hormones facilitating both 17α-hydroxylase and 17,20-lyase activities. We characterized a partial CYP17A1 deletion in a Kurdish family with 17OHD by multiplex ligation-dependent probe amplification (MLPA). The index patient presented with amenorrhea and lack of pubertal development. Investigations established the diagnosis of 46,XY disorder of sex development (DSD). She is the daughter of consanguineous parents and has 2 sisters with similar clinical presentation. All patients showed biochemical signs of primary adrenal and gonadal insufficiency. The molecular genetic analysis by PCR suggested a deletion spanning exons 1-6 of the CYP17A1 gene. MLPA analysis confirmed the large partial CYP17A1 deletion in patients and parents in homozygous and heterozygous state, respectively. This is the first report employing MLPA for mutation analysis to detect a deletion of CYP17A1 spanning multiple exons in 3 patients with classic 17OHD. Therefore, it is important to consider large partial CYP17A1 deletions in 17OHD in addition to point mutations in cases where no segregation analysis is possible to determine the correct genotype.