MALE NEOGAMETOGENESIS TO GENERATE FULLY DEVELOPED PREIMPLANTATION EMBRYOS

Abstract

To assess the feasibility of haploidizing epiblast-like cells (EpiLCs) derived from male mouse embryonic stem cells (mESCs) to generate full preimplantation embryos. Male mESCs were propagated and differentiated in a novel 3D culture system by the direct spherification technique. After 3 days of exposure to differentiating medium, EpiLCs, confirmed by specific biomarkers and synchronized at G1, were fused with intact oocytes following activation and completion of biparental haploidization. Full preimplantation development of the zygotes was assessed by time-lapse imaging and compared to control ICSI conceptuses. Male mESCs were suspended in base spherification solution and encapsulated by exposure to sodium alginate. Each sphere containing approximately 6.0x10⁵ cells, with diameter ranging from 8 to 10 mm, was bathed in medium supplied with activin A, bFGF, and KSR. Differentiated EpiLCs were isolated by trypsinization to assess OCT4 and Nanog expression. An additional 1.5 μM aphidicolin was supplied to the media 24 h before fusing with oocytes. Individual EpiLCs treated by Sendai virus were injected in the perivitelline space of an oocyte. Resulting oocytes displaying 2 spindle complexes were activated by 10 μM calcium ionophore. Control embryos were generated by piezo-actuated ICSI. Embryonic developmental morphokinetic parameters were detected through time-lapse imaging. Male mESCs, engulfed in 4 spheres and plunged in differentiating medium, aggregated into embryoid bodies with a mean diameter of 250 μm. After isolation by mechanically breaching the spheres followed by trypsinization, cells showed a positive expression of OCT4 (>90%) and a decreased Nanog positivity (<40%), confirming differentiation to EpiLCs. Sendai virus–mediated cell fusion was performed on 80 oocytes, with a 100% fusion rate. A total of 49 oocytes displayed biparental spindle complexes (61.3%). After oocyte activation, 37 zygotes extruded the second polar body alongside a third pseudo polar body (from the haploidized EpiLCs), and developed 2 pronuclei (75.5%). After 96 h in a time-lapse incubator, the control embryos developed into 2-cell (86.7%), 4-cell (83.3%), morula (83.3%) and blastocyst (83.3%) embryos. Meanwhile, embryos in the experimental group developed into 2-cell embryos at a lower rate (69.4%, P < 0.001). Further developments into 4-cell (49.0%), morula (32.7%) and blastocyst (32.7%) embryos were also lower in zygotes obtained from biparental haploidization (P < 0.00001). Neogametogenesis was accomplished by differentiating mESCs to EpiLCs in a novel 3D culture system. Zygotes obtained from biparental haploidization yielded full preimplantation development to expanded/hatching blastocysts. Despite a lower rate of blastocysts development, embryo morphokinetic characteristics were comparable to the control ICSI conceptuses. Once the ability to generate healthy pups is confirmed, this model may represent an ideal option to treat men with germ cell aplasia or spermatogenic arrest.