Ex Vivo Spatiotemporal Characterization of Spermatogenesis in Mouse Testicular Organoids

Testicular organoids (TOs) offer an opportunity to preserve fertility, but current TO protocols are limited by suboptimal maintenance of the organoid structure, meiotic defects, and incomplete in vitro spermatogenesis (IVS). Here, a strategy is developed for self-reconstitution of single-cell suspensions of neonatal mouse testes into TOs containing the major testicular cell types and generation of tubular-like structures and haploid spermatids. Morphological and lineage-specific 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-4/5-like round spermatids in vivo. Further scRNA-seq analysis confirmed that spermatid-like cells accounted for ≈2.43% 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-based findings provide a promising platform for studies on testicular microenvironment construction, IVS, and fertility preservation.