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EFFECTS OF THE STAGE OF CELL CYCLE OF DONOR NUCLEI AND ACTIVATION OF RECIPIENT OOCYTES ON DEVELOPMENT OF RECONSTRUCTED BOVINE EMBRYOS.
Sawai, Ken1, Moriyasu, Satoru 1, Minamihashi, Akira1, Kageyama, Soichi1, Hirayama, Hiroki1, Ashino, Masashiro1, Kitano, Noriyasu1, Onoe, Sadao1, 1
ABSTRACT- The stage of cell cycle of donor nuclei and activation of recipient oocytes are important factors for reprogramming of the nuclei and development of reconstructed embryos. The present study was to examine the effects of these factors on development of reconstructed bovine embryos. Recipient oocytes matured in culture were enucleated and some enucleated oocytes were activated by treatment with Ca-ionophore and cycloheximide (CH). Non-activated oocytes were treated with CH after fusing with a donor nucleus. In Experiment I, bovine fetal fibroblast (BFF) cells were cultured in medium containing 10% (normal)or 0.5% (serum starvation) fetal bovine serum and fused with recipient oocytes. Most BFF cells (normal, 85%; serum starvation, 96%)cultured in both conditions were in G0-G1 phase of cell cycle. No difference was detected in blastocyst formation between oocytes fused with BFF cells cultured in different conditions. More blastocysts were formed in non-activated (18-33%) than activated (3-6%) oocytes (P < 0.05) irrespective of culture condition of BFF cells transferred. In Experiment II, bovine embryonic cells from the morula stage that are known as in S phase were fused with recipient oocytes. In contrast with the results in BFF cells, blastocyst formation was higher in activated (39%) than non-activated (8%) oocytes (P < 0.05). In Experiment III, it was examined whether fusion of BFF cells at S phase with activated oocytes stimulates blastocyst formation. The cell cycle of BFF cells was synchronized with G0, late G1 or S phase by culturing the cells in a medium with or without aphidicolin. When these cells were fused with activated or non-activated oocytes, 73-87% of the reconstructed oocytes cleaved to the 2-8-cell stage. However, blastocyst formation (23%) was obtained only in non-activated oocytes fused with BFF cells at G0 phase. These results suggest that the mechanism(s) by which donor nuclei are initialized in the recipient oocytes is different between somatic and embryonic cells.
KEY WORDS: nuclear transfer, cell cycle, oocyte activation, somatic cells
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