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Here, a strategy is developed for self\u2010reconstitution of single\u2010cell suspensions of neonatal mouse testes into TOs containing the major testicular cell types and generation of tubular\u2010like structures and haploid spermatids. Morphological and lineage\u2010specific marker analyses revealed that these TOs met the criteria for organoids, including germ cell differentiation and recapitulation of key events in meiosis (e.g., chromosome recombination and synapsis). Notably, the spatiotemporal characteristics of spermatogenesis in the TOs are comparable to those in testes, and their derived haploids resembled step\u20104\/5\u2010like round spermatids in vivo. Further scRNA\u2010seq analysis confirmed that spermatid\u2010like cells accounted for \u22482.43% of the total germ cells. Next, undifferentiated germ cells are able to develop into haploid spermatids even when chimeric TOs are reconstructed via testicular somatic cells (Sertoli cells or Leydig cells) from mice with different genetic backgrounds. Collectively, the TO\u2010based findings provide a promising platform for studies on testicular microenvironment construction, IVS, and fertility preservation.<\/jats:p>","DOI":"10.1002\/advs.202512670","type":"journal-article","created":{"date-parts":[[2025,11,1]],"date-time":"2025-11-01T03:10:27Z","timestamp":1761966627000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Ex Vivo Spatiotemporal Characterization of Spermatogenesis in Mouse Testicular Organoids"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3870-8541","authenticated-orcid":false,"given":"Jiachen","family":"Sun","sequence":"first","affiliation":[{"name":"State Key Laboratory of Reproductive Medicine and Offspring Health Nanjing Medical University  Nanjing Jiangsu 211166 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