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, we developed a strategy for self-reconstitution of single-cell suspensions of neonatal mouse testes into TOs containing the major testicular cell types and generation of lumen-like structures and haploids. 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 were comparable to those in testes, and their derived haploids resembled step-4/5-like round spermatids in vivo. Further scRNA-seq analysis confirmed that haploids accounted for approximately 2.43% of the total germ cells and that undifferentiated germ cells were able to develop into haploids even when chimeric TOs were reconstructed via testicular somatic cells (Sertoli cells or Leydig cells) from mice with different genetic backgrounds. Collectively, our TO-based findings provide a promising platform for studies on testicular microenvironment construction, IVS and fertility preservation. Overall design: Cells were isolated through enzymatic dissociation from both 28-day-old testicular organoids (TOs) and 28-day-old mouse testes.