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Sertoli-Sertoli-germ cell tight and adherent junctions may play an essential role in spermatogonial stem cells (SSCs) to differentiate spermatocytes and functional haploid spermatozoa.In this study, we have developed a new SSC culture system with Sertoli cells that could derive spermatocytes and haploid cells from SSCs.We observed that the in vitro derived spermatocytes could regulate the relevant tight and adherent junctions;We have provided sufficient evidence to show that the Sertoli cells play a significant role in contributing to the mature haploid cells derived from the cultured SSCs.These results suggest that in vitro induced haploid cells from SSCs structurally and nutritionally rely on the Sertoli cell supports for their development and differentiation through theft cell junctions.This in vitro system could not only facilitate the exploration of genes involved in SSCs differentiation and may also discover potential targets used to perturb spermatogenesis for developing a novel male contraceptive.