NRG1 and KITL signal downstream of retinoic acid in the germline to support soma-free syncytial growth of differentiating spermatogonia

Defined culture systems supporting spermatogonial differentiation will provide experimental platforms to study spermatogenesis. However, germline-intrinsic signaling mechanisms sufficient to support spermatogonial differentiation without somatic cells remain largely undefined. Here we analyzed EGF superfamily receptor and ligand diversity in rat testis cells and delineated germlineintrinsic signaling via an ERBB3 co-transducer, ERBB2, as essential for retinoic acid-induced syncytial growth by differentiating spermatogonia. Similar to the ERBB2/3 agonist NRG1, we found that KIT Ligand (KITL) robustly supported spermatogonial differentiation without serum or somatic cells. ERBB2 inhibitors failed to disrupt KITL-dependent spermatogonial development, and KITL prevented ERBB3-deficient spermatogonial degeneration upon differentiation. Thus we report that NRG1 and KITL activate alternative pathways downstream of retinoic acid signaling in the germline that are essential for stem cells to undergo premeiotic steps of spermatogenesis in culture. Robust serum/soma-free spermatogonial differentiation opens new doors to study mammalian germ cell biology in culture and to discover factors that can drive meiotic progression in vitro.