Embryonic Stem Cell Lines Derived from Somatic Cell Nuclear Transferred Oocytes Fertilizing with Sperm

Metaphase II (MII) oocyte can halve its genome by extrusion of the second polar body at fertilization. We aim to confirm the ability of MII ooplasm to reprogram and haploidyze somatic cell nucleus. We tested, by somatic cell nuclear transfer, the ability to haploidyze mouse cumulus cell nuclei. Generated constructs were then subjected to intracytoplasmic sperm injection (ICSI) to generate embryos and derive embryonic stem cells (ESCs). MII oocytes were recovered from female mice 16-18h after hCG injection. Superovulation was achieved by injecting PMSG (10IU/mouse) followed by injection of hCG (10IU/mouse) 48h later. H3.3B-HA-iEYFP or tdTomato cumulus cells were used for nuclear transfer, and sperm carrying a different fluorescence reporter was used for ICSI to activate the constructed oocytes. After extrusion of pseudo-polar bodies (psPBs) and fertilization assessment, eventual morulae and blastocysts were used to derive ESCs. psPBs extrusions were observed in 51% (80/154) of the reconstructed oocytes 2-3h after fertilization. Immunofluorescence (IF) staining revealed that bipolar spindle around the cumulus nucleus was visible 1-3h following fertilization and psPBs extrusion. Using H3.3B-HA-iEYFP (chromosome 17) heterozygous cumulus cells, we found that half of the embryos were EYFP positive, indicating proper somatic nuclear haploidization in these embryos. In a total of 118 tdTomato nuclear-transferred oocytes, 55 (47%) of them extruded psPBs at fertilization. In these 55 psPB embryos, 48 (87%) of them reached the 2-cell stage, 42 (76%) the 4-cell stage, 23 (42%) the morula stage, and 6 (11%) the early blastocyst stage. The 6 early blastocysts were used for ESC derivation and one line was established (OIH-td-cum-1). Of the 63 oocytes that did not extrude psPBs, 41(65%), 30(48%), 17(27%) and 10(16%) embryos developed to 2-cell, 4-cell, morula and early blastocyst stage, respectively. Two ESC lines were derived from the blastocysts (OIH-td-cum-2, OIH-td-cum-3). All 3 of the ESC lines were tdTomato positive, maintained typical ESC morphology, and expressed pluripotency markers. Karyotyping analysis showed that ESC line OIH-td-cum-1 was a diploid karyotype with a gain of chromosomes 5, 8, 9, 18, and 19. OIH-td-cum-2 and OIH-td-cum-3 were triploid karyotypes (60 chromosomes). Somatic genome DNA can be reduced in the MII oocyte cytoplasm through the extrusion of pseudo-polar bodies after fertilization. Embryos containing haploidyzed somatic genome and sperm DNA can develop into blastocysts and derive ESC lines. Haploidization of diploid nuclei in MII oocytes would be an alternative approach to producing artificial gametes.