Induction of Male Neogametogenesis in a Three-Dimensional Microenvironment Supporting Successful Fertilization and Proper Embryo Development

In regenerative medicine, several attempts have been made to produce functional de novo gametes from mouse embryonic stem cells by utilizing 3D culture systems. We attempted to perform neogametogensis in a novel 3D niche to generate neo-gametes ready to be used for insemination. Mouse embryonic stem cells (ESCs) were initially cultured on a gelatin-coated 6-well plate with a monolayer of fibroblasts before being encapsulated in sodium alginate spheres. These spheres were then immersed in specially formulated epiblast like-cell (EpiLC) followed by primordial germ cell-like cell (PGCLC) medium to promote differentiation into germ-like cells. Over the course of differentiation, immunofluorescence analysis revealed consistent expression patterns of spermatogenic markers. Cells were assessed for DAZL (early germ cell marker) VASA (spermatocyte), BOULE (post-meiotic) and acrosin (spermatid). The differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored via time-lapse microscopy. Spherified neogametes on D(day)22, 29, and 36 achieved fertilization rates of 61.1%, 82.7%, and 80.0%, respectively, and blastulation rates of 20.5%, 36.0%, 26.3%, respectively. Controls exhibited fertilization rates of 89.1% and blastulation rates of 76.3%. When embryo morphokinetics were considered, D29 embryos reached compaction at 64 h and blastulation at 77 h, mirroring closely the control's timing of 63 h and 77 h, respectively. D 22 embryos displayed the most delayed embryo development, of which compaction occurred at 68 h and blastulation at 106 h. Despite normal fertilization and successful blastulation, the efficiency rate remained below optimal levels. Nevertheless, our system could produce viable offspring, demonstrating that replicating the three-dimensional seminiferous tubule environment is crucial for generating artificial gametes. This eliminates the need for allo-/xeno-genic transplantation in experimental animals.