INFLUENCE OF RECIPIENT OOCYTE CELL-CYCLE STAGE ON DNA-SYNTHESIS, NUCLEAR-ENVELOPE BREAKDOWN, CHROMOSOME CONSTITUTION, AND DEVELOPMENT IN NUCLEAR TRANSPLANT BOVINE EMBRYOS

Nuclear transplantations into metaphase II (MII) and S phase oocyte cy toplasm were performed to investigate the influence of recipient cell cycle stage on nuclear function and development of bovine nuclear tran splant (NT) embryos. Rate of inactivation of histone H1 kinase and dur ation of DNA synthesis in activated oocytes were determined. The propo rtion of S phase blastomeres in in vivo produced day 5.5 bovine embryo s was measured. DNA synthesis was also assessed in NT embryos after tr ansfer into MII and S phase cytoplasm. MII NT embryos were produced by fusing a blastomere into a MII oocyte; the fusion pulse served to act ivate the oocyte. S NT embryos were produced by fusing a blastomere in to an early S phase oocyte electrically activated 4 h prior to fusion. Nuclear envelope structure, chromosome constitution, and extent of de velopment were examined in MII and S NT embryos. Histone H1 kinase act ivity dropped to baseline within 2 h of electrical activation. A secon d 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 H-3-thymidine. Nuclear transplantation into MII cytoplasm resulted in a transient drop in DNA synthesis over 3.5 h. DNA synthesi s resumed at 4.5 h post activation (hpa), concomittantly with initiati on of DNA replication in activated oocytes. In contrast, DNA synthesis was not interrupted after transfer into S phase cytoplasm. DNA synthe sis persisted until 13.5 hpa, as in activated oocytes. Partial or comp lete nuclear envelope breakdown (NEBD) occurred after transfer into MI I cytoplasm, whereas the nuclear envelope remained intact in 50% of th e embryos or underwent partial breakdown in S phase cytoplasm. A great er 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 m orula or blastocyst stage (22% vs. 5%, P < 0.001). The data indicate t hat DNA synthesis is regulated differently if the recipient oocyte is in MII or in S phase at the time of fusion. Extended DNA synthesis aft er transfer into MII cytoplasm suggests a re-replication of the donor chromatin. Re-replication, presumably, does not occur after transfer i nto S phase cytoplasm. Re-replication is likely to be a consequence of permeabilization of the nuclear envelope upon NEBD in MII cytoplasm. Improved regulatien of DNA synthesis after transfer into S phase cytop lasm and reduced incidence of chromosome damage in the first cell cycl e may have been responsible for increased frequency of development of S NT embryos to the morula/blastocyst stage. (C) 1993 Wiley-Liss, Inc.