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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Genmark, Inc., Salt Lake City, Utah 84108.
F L
FL
Barnes
P
P
Collas
R
R
Powell
W A
WA
King
M
M
Westhusin
D
D
Shepherd
D000818:Animals; D002417:Cattle; D002453:Cell Cycle; D002467:Cell Nucleus / Q000648:ultrastructure; D003593:Cytoplasm / Q000648:ultrastructure; D004247:DNA / Q000096:biosynthesis; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005314:Embryonic and Fetal Development; D066298:In Vitro Techniques; D007621:Karyotyping; D008854:Microscopy, Electron; D009685:Nuclear Envelope / Q000648:ultrastructure; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D006656:Protamine Kinase / Q000378:metabolism
D004247:DNA; D006656:Protamine Kinase