Endocrine Abstracts

Zebrafish has emerged as an important vertebrate in vivo model to study human disease. Steroidogenesis in zebrafish is not well characterised. Human CYP11A1 (hCYP11A1) catalyses the first step of steroidogenesis, the conversion of cholesterol to pregnenolone. Zebrafish Cyp11a1 (zCyp11a1) is essential during embryogenesis. Published data suggest that zCyp11a1 facilitates steroidogenesis in the interrenal (equivalent to mammalian adrenal), gonad and brain. We identified a duplicated gene, designated as zCyp11a2, sharing 85% sequence identity with zCyp11a1.

The aim of this study was to characterise the zCyp11a paralogs gene expression pattern and function.

Our RT-PCR data shows that zCyp11a1 is expressed during early development, from 0 to 22 h post-fertilisation (hpf). Conversely, zCyp11a2 was only detected after the interrenal is formed (from 32 hpf). Adult gonads expressed both paralogs. The interrenal and brain expressed only zCyp11a2. The in vivo zCyp11a function was assessed by knockdown studies using zCyp11a-morpholinos. Pregnenolone and cortisol were measured in the developing zCyp11a morphants by liquid chromatography/tandem mass spectrometry (LC/MS/MS). zCyp11a1 morphants showed an abnormal early development and reduced pregnenolone levels. Transient zCyp11a2 knockdown impaired de novo pregnenolone and cortisol synthesis and resulted in a late phenotype indicative of metabolic abnormalities. zCyp11a enzyme activity was assessed in COS7 cells transiently co-overexpressing adrenodoxin and zCyp11a or hCYP11A1. Cells were incubated with 22R-hydroxycholesterol and pregnenolone was measured by LC/MS/MS. zCyp11a2 activity was similar to hCYP11A1, whilst zCyp11a1 activity was significantly reduced.

Our data completely revises our understanding of zebrafish steroidogenesis by defining the roles of the previously described zCyp11a1 and the newly discovered zCyp11a2 enzyme. zCyp11a2 rather than zCyp11a1 is the hCYP11A1 ortholog in zebrafish steroidogenic tissues. Furthermore, we prove that zCyp11a1 activity is significantly reduced but essential during embryogenesis. Importantly, this study proves the value of zebrafish as a comprehensive in vivo model in translational research of adrenal and gonadal disease.

Declaration of funding: The project was supported by a Wellcome Trust ISSF grant (to N K), a Society for Endocrinology early career grant (to S P) and a Marie Curie Intra-European Fellowship (grant number IEF-GA-2009-255424, 2009 to S P)