Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the cell cycle in granulosa and fibroblast cells of cattle.
The cell cycle stage of donor cells is an important factor influencing developmental ability of nuclear transfer embryos. In the present experiment, cumulus and fibroblast cells of cattle were subjected to flow cytometric cell cycle analysis before being used in somatic cloning experiments. The following experimental groups were analyzed for each cell type: (1) actively dividing cells, (2) cells confluent for 4 days, (3) cells starved for 1, 2, 3 or 5 days. Using the propidium iodide flow cytometric assay, there were no significant differences (P > or = 0.05) in the percentage of cells in G0/G1 regardless of origin and type of cell, after confluency or serum starvation. Differences with the growing cells were found (P < or = 0.01). To determine what subset of cells in G0/G1 were in the G0 subphase of the cell cycle, an immunofluorescence analysis was conducted using monoclonal anti-PCNA antibodies in a FACS assay. There were not statistically significant differences in the percentage of cells that enter G0, between confluent and any starved group for either type of cells. Bovine fibroblast cells, confluent or serum starved for 3 days, were used in nuclear transfer experiments. A slight trend for a more desirable fusion rate in starved cells was detected, and embryo cleavage was greater in starved cells, however, in vitro development to blastocysts was similar between groups. Data indicate that prolonged culture of cells in the absence of serum does not imply a shift in the percentage of cells that enter G0/G1 or G0 alone, and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs, because there are negative effects such as apoptosis, associated with serum starvation.
Hayes
O
O
Division of Animal Biotechnology, Centro de Ingeniería Genética y Biotecnología, Havana 10600, Cuba.
Ramos
B
B
Rodríguez
L L
LL
Aguilar
A
A
Badía
T
T
Castro
F O
FO
D000818:Animals; D002417:Cattle / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology; D016895:Culture Media, Serum-Free; D004247:DNA / Q000502:physiology; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D005347:Fibroblasts / Q000166:cytology*; D005434:Flow Cytometry; D006107:Granulosa Cells / Q000166:cytology*
D016895:Culture Media, Serum-Free; D004247:DNA