Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution, and development in nuclear transplant bovine embryos

Abstract

Nuclear transplantations into metaphase II (MII) and S phase oocyte cytoplasm were performed to investigate the influence of recipient cell cycle stage on nuclear function and development of bovine nuclear transplant (NT) embryos. Rate of inactivation of histone H1 kinase and duration of DNA synthesis in activated oocytes were determined. The proportion of S phase blastomeres in in vivo produced day 5.5 bovine embryos was measured. DNA synthesis was also assessed in NT embryos after transfer into MII and S phase cytoplasm. MII NT embryos were produced by fusing a blastomere into a MII oocyte; the fusion pulse served to activate the oocyte. S NT embryos were produced by fusing a blastomere into an early S phase oocyte electrically activated 4 h prior to fusion. Nuclear envelope structure, chromosome constitution, and extent of development were examined in MII and S NT embryos. Histone H1 kinase activity dropped to baseline within 2 h of electrical activation. A second electrical pulse did not alter H1 kinase activity when delivered 4 h after the first pulse. The frequency of S phase blastomeres in day 5.5 bovine embryos ranged from. 79% to 100%, depending on the duration of culture in ³H-thymidine. Nuclear transplantation into MII cytoplasm resulted in a transient drop in DNA synthesis over 3.5 h. DNA synthesis resumed at 4.5 h post activation (hpa), concomittantly with initiation of DNA replication in activated oocytes. In contrast, DNA synthesis was not interrupted after transfer into S phase cytoplasm. DNA synthesis persisted until 13.5 hpa, as in activated oocytes. Partial or complete nuclear envelope breakdown (NEBD) occurred after transfer into MII cytoplasm, whereas the nuclear envelope remained intact in 50% of the embryos or underwent partial breakdown in S phase cytoplasm. A greater proportion of S NT embryos was diploid (50% vs. 23% MII NT embryos, P < 0.001), and a higher frequency of S NT embryos developed to the morula or blastocyst stage (22% vs. 5%, P < 0.001). The data indicate that DNA synthesis is regulated differently if the recipient oocyte is in MII or in S phase at the time of fusion. Extended DNA synthesis after transfer into MII cytoplasm suggests a re-replication of the donor chromatin. Re-replication, presumably, does not occur after transfer into S phase cytoplasm. Re-replication is likely to be a consequence of permeabilization of the nuclear envelope upon NEBD in MII cytoplasm. Improved regulation of DNA synthesis after transfer into S phase cytoplasm and reduced incidence of chromosome damage in the first cell cycle may have been responsible for increased frequency of development of S NT embryos to the morula/blastocyst stage.