Impaired androgen activity induces defective sexual differentiation of the male reproductive tract, including hypospadias, an abnormal formation of the penile urethra. Androgen signaling in the urethral mesenchyme cells (UMCs) plays essential roles in driving dimorphic urethral development. However, cellular events for sexual differentiation remain virtually unknown. In this study, histological analyses, fluorescent staining, and transmission electron microscopy (TEM) were performed to reveal the cellular dimorphisms of UMCs. F-actin dynamics and migratory behaviors of UMCs were further analyzed by time-lapse imaging. We observed a prominent accumulation of F-actin with poorly assembled extracellular matrix (ECM) in female UMCs. In contrast, thin fibrils of F-actin co-aligning with the ECM through membrane receptors were identified in male UMCs. Processes for dimorphic F-actin assemblies were temporally identified during an androgen-regulated masculinization programming window and spatially distributed in several embryonic reproductive tissues. Stage-dependent modulation of the F-actin sexual patterns by androgen in UMCs was also demonstrated by time-lapse analysis. Moreover, androgen regulates coordinated migration of UMCs. These results suggest that androgen signaling regulates the assembly of F-actin from cytoplasmic accumulation to membranous fibrils. Such alteration appears to promote the ECM assembly and the mobility of UMCs, contributing to male type genital organogenesis. (C) 2017 S. Karger AG, Basel