Testosterone acts through the membrane protein GPRC6A to cause cardiac edema in zebrafish embryos

Androgens are classically thought to act through intracellular androgen receptors (AR/NR3C4), but they can also trigger non-genomic effects via membrane proteins. Although several membrane androgen receptors have been characterized in vitro, their functions in vivo remain unclear. Using a chemical-genetic screen in zebrafish, we found that GPRC6A, a G-protein-coupled receptor, mediates non-genomic androgen actions during embryonic development. Exposure to androgens (androstanedione, DHT and testosterone) caused cardiac edema or tail curvature in wild-type embryos, as well as in ar mutants, suggesting AR-independent pathways. We then mutated putative membrane androgen receptors [gprc6a, hcar1-4 and zip9 (slc39a9)] and found that only gprc6a mutants exhibited a significant reduction in cardiac edema after testosterone exposure. Additionally, co-treatment of wild-type embryos with testosterone and GPRC6A antagonists significantly suppressed the cardiac edema phenotype. Using RNA-seq and RNA rescue approaches, we found that testosterone and GPRC6A cause cardiac phenotypes by reducing Pak1 signaling. Our results indicate that testosterone induces cardiac edema in zebrafish embryos through GPRC6A, independent of nuclear androgen receptors, highlighting a previously unappreciated non-genomic androgen signaling pathway in embryonic development.