Bovine Nuclear Transfer Using Primary Cultured Cumulus Cells of Determined Cell Cycle-Phase

摘要

Normal development of nuclear transfer embryos is thought to be dependent on transferal of nuclei in GO or G1 phases of the cell cycle. Therefore, the objective of this study was to investigate the cell cycle phase of different sizes of bovine cumulus cells cultured under serum starvation conditions and the consequent development after nuclear transfer. Cumulus cells after oocyte maturation were separated and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS) at 37°C, 5% CO2 in air for 3-4 days followed by another 3-4 days under the same conditions but with 0.5% FCS (serum starvation culture). Percentages of cells in the various stages of the cell cycle were calculated using flow cytometry with gates were set to include only small, medium or large-sized cells. Gating for different cell sizes was guided by microscopic measurement of trypsinised cell population. Cells of different designated sizes were electrically fused to metaphase II arrested enucleated oocytes. Reconstituted embryos were cultured in modified synthetic oviduct fluid medium supplemented with amino acids, glucose, insulin and BSA at 39°C, 5% CO2, 5% O2 and 90% N2. Development to blastocysts and cell count were checked 174 h post-fusion. Flow cytometric analysis revealed that small and medium-sized cells had significantly higher percentages (p< 0.05) of nuclei existing in the G0/G1 phase (93-94%) than large cells (83%). Serum-starved cells of different sizes fused at the same rate to the enucleated oocytes. No significant difference (p>0.05) was observed in cleavage, development to blastocyst and blastocysts cell number after embryos reconstruction from different cell sizes. In conclusion the present study showed that different sizes of the primary cultured, serum starved cumulus cells can be used as donor nuclei at the G/0G1-phase.