Testosterone acts through membrane protein GPRC6A to cause cardiac edema in zebrafish emrbyos
Classically, intracellular nuclear androgen receptors (ar) are considered to be the key mediators of androgen actions. However, there have been several reports that androgens can also exert their effects by binding to integral membrane proteins. Despite in vitro cloning and characterization of several putative membrane androgen receptors, their functions in vivo remain poorly understood. We used a chemical-genetic screen in zebrafish and discovered that the G-protein coupled receptor, GPRC6A, mediates androgen action during embryonic development. We exposed zebrafish embryos to testosterone (30 µM) from 2-4 hours post-fertilization (hpf) until 72 hpf. We found that testosterone exposure caused cardiac edema in wild-type and ar mutant zebrafish embryos, but there was a significant reduction in this phenotype in the grprc6a mutant embryos, suggesting that gprc6a regulates androgen action independently of nuclear androgen receptors. Additionally, we exposed wild-type embryos to testosterone together with GPRC6A antagonists and observed a significant suppression of the cardiac edema phenotype. These results suggest that testosterone causes cardiac edema in zebrafish embryos by acting via the integral membrane protein GPRC6A, independently of nuclear androgen receptors. Overall, our study provides insights into non-genomic androgen signaling during embryonic development and identifies GPRC6A as a key receptor mediating androgen action. Overall design: To identify gene targets of Testosterone-GPRC6A, we performed RNAseq on 3 day post fertilization zebrafish embryos following 3 day exposure to either: testosterone, testosterone + strong GPRC6A antagonist EGCG, testosterone + weak GPRC6A antagonist calindol, 0.1% DMSO (vehicle control). We then performed gene expression analysis comparing each chemical exposure to vehicle with 3 biological replicates for each condition.