The development of mouse zygotes after fusion with synchronous and asynchronous cytoplasm

Cytoplasts with diameters of 40-45, 50-55 and 70-75 microm, derived from mouse oocytes at the germinal vesicle, metaphase II and zygote stages were incorporated into zygotes by electrofusion. Manipulated (n = 867) and culture-control (n = 1114) embryos were cultured in vitro and transferred to pseudo-pregnant recipients at the blastocyst stage. When synchronous cytoplasts measuring 40-45 and 50-55 microm in diameter were incorporated into 138 and 86 zygotes respectively, only one embryo in each group (not significant) became arrested at the 1-cell stage. A total of 124 (89.9 compared with 91. 6% for controls) and 69 embryos (80%, P < 0.001 compared with 91.6% for controls) reached the blastocyst stage respectively. In the first group, 66 out of 106 blastocysts implanted (62.2 compared with 54.9% for controls; not significant), however, only 24 (22.6 compared with 40.2% for controls, P < 0.001) were viable in comparison with controls. There were four groups of zygotes that received metaphase II cytoplasts. In the first group, 200 zygotes were fused with 40-45 microm cytoplasts. The second group of 145 zygotes were fused with cytoplasts of the same size derived from aged oocytes. In the third and fourth groups, 38 and 36 zygotes were fused with 50-55 and 70-75 microm cytoplasts respectively. In the first two groups, none of the embryos arrested at the 1-cell stage, but in the other groups the rates were 15 out of 38 (39.5%) and 36 out of 36 (100%) respectively. These zygotes remained arrested at the pronuclear stage and contained large inflated pronuclei. The blastocyst formation rates were 183 out of 200 (91.5 compared with 91.6% for controls, not significant), 109 out of 145 (75.2% lower than controls, P < 0.05) and 14 out of 38 (39.5% lower than controls, P < 0.0001) respectively. In the first two groups 109 and 25 blastocysts were transferred, of which 76 (69.7%) and 15 (60.0%) implanted. This was higher than control embryos (54.9%, P < 0.01) for the first group and similar to controls for the second group. In the first group, 60 embryos (55%) were viable on day 10 of transfer in comparison with controls (40.2%, P < 0.05) while in the second group, 11 embryos (44.0%, not significant) were viable on day 10 of transfer. Zygotes that received germinal vesicle stage cytoplasts developed poorly and the implantation rate was significantly reduced. The present study confirms the importance of the ooplasmic domain in meiotic maturation and preimplantation development. Our results suggest that implantation may be enhanced by transfer of a small amount of metaphase II cytoplasm to the mouse embryo during the 1-cell stage; however, fusion of intact zygotes with cytoplasts > 45 microm appeared largely detrimental. The mechanisms responsible for these changes are yet unknown.