Induction of male neogametogenesis in a three-dimensional microenvironment supporting successful fertilization and...

Area of Science:

• Regenerative Medicine
• Developmental Biology
• Stem Cell Biology

Background:

• Generating functional gametes in vitro is a key goal in regenerative medicine.
• Previous attempts using 3D culture systems have shown promise but require optimization.
• Mouse embryonic stem cells (ESCs) offer a viable source for in vitro gamete production.

Purpose of the Study:

• To develop a novel 3D niche for neogametogenesis from mouse ESCs.
• To generate artificial gametes suitable for insemination and subsequent embryo development.
• To assess the efficiency and viability of gametes produced in the 3D system.

Main Methods:

• Mouse ESCs were cultured and encapsulated in sodium alginate spheres.
• Cells were differentiated using epiblast-like cell (EpiLC) and primordial germ cell-like cell (PGCLC) media.
• Immunofluorescence analysis confirmed expression of key spermatogenic markers (DAZL, VASA, BOULE, acrosin).
• Differentiated cells were injected into oocytes, activated, and embryo development was monitored.

Main Results:

• Neogametes produced on days 22, 29, and 36 achieved fertilization rates of 61.1%, 82.7%, and 80.0%, respectively.
• Blastulation rates reached 20.5%, 36.0%, and 26.3% for days 22, 29, and 36, respectively.
• Embryo morphokinetics for D29 neogametes closely mirrored control timings, indicating successful development.
• Viable offspring were produced, though overall efficiency remained below optimal levels.

Conclusions:

• Replicating the three-dimensional seminiferous tubule environment is critical for generating functional artificial gametes.
• The developed 3D culture system shows potential for producing gametes without allo-/xeno-genic transplantation.
• Further optimization is needed to improve the efficiency of neogametogenesis for clinical applications.