ABSTRACT
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.
Footnotes
Author contributions
Conceptualization: V.Z., D.A.G.; Data curation: S.S.; Formal analysis: S.S., D.A.G.; Funding acquisition: D.A.G.; Investigation: V.Z., S.S., D.A.G.; Methodology: V.Z., S.S.; Project administration: D.A.G.; Supervision: D.A.G.; Visualization: D.A.G.; Writing – original draft: V.Z., D.A.G.; Writing – review & editing: V.Z., D.A.G.
Funding
This work was supported by the National Institutes of Health Training Program in Precision Environmental Health Sciences (T32 ES027801). Deposited in PMC for release after 12 months.
Data availability
All RNA-seq data have been deposited in GEO under accession number GSE274852.
The people behind the papers
This article has an associated ‘The people behind the papers’ interview with some of the authors.
