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
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 EpiLC
followed by 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 D22, 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 taken in
consideration, D29 embryos reached compaction at 64h and blastulation at
77h, mirroring closely the control’s timing of 63h and 77h, respectively.
D22 embryos displayed the most delayed embryo development, of which
compaction occurred at 68h and blastulation at 106h. 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.