Germ cells are essential for testicular morphogenesis and functional reconstruction in a porcine xenograft model.

Reconstruction of functional testis tissue can provide new opportunities for in vitro spermatogenesis and fertility restoration in livestock. Although testis reconstruction has been reported in several species, the germ cell dependent mechanisms underlying de novo testicular morphogenesis remain insufficiently defined in large animals. In this study, we investigated the contribution of porcine spermatogonial stem cells (pSSCs) to testis morphogenesis and spermatogenesis using a xenograft model. Testicular cells containing approximately 10% germ cells combined with a somatic-enriched fraction (SEF) (GF group) or SEF only (FO group) were transplanted subcutaneously into immunodeficient mice and examined after six months. GF tissues developed well-organized seminiferous tubules and showed multiple spermatogenic stages, whereas FO tissues remained fragmented, poorly organized, and lacked stable tubular architecture. Immunofluorescence analyses revealed appropriate localization of germ cells and maturation-associated features of Sertoli cells in GF tissues, whereas FO tissues exhibited aberrant Sertoli cell localization and marker expression patterns. Transcriptomic profiling further showed enrichment of cell cycle– and spermatogenesis-related pathways in GF tissues, while FO tissues exhibited upregulation of coagulation-, inflammation-, and early developmental pathways, consistent with impaired tissue organization. Collectively, these findings demonstrate that pSSCs are indispensable for initiating and stabilizing testicular morphogenesis in xenograft condition and underscore the utility of this model for advancing reproductive biotechnology in livestock.