53 CELL CYCLE SYNCHRONIZATION OF DONOR CELLS AT G1 PHASE AND DEVELOPMENTAL ABILITY OF NUCLEAR TRANSFER EMBRYOS IN MINIATURE PIGS

Abstract

The cell cycle of donor cells is one of the essential factors for the success of somatic cell nuclear transfer (SCNT), and G0/G1-phase cells have been widely used as donor cells. However, cells synchronized at the G0/G1-phase also have a population of cells with cell cycles other than G0/G1-phase, and we cannot precisely know the cell cycle of donor cells being used for SCNT. In this experiment, we reconstructed SCNT embryos from donor cells that were synchronized at the G1-phase or at the G0/G1-phase and compared their developmental ability in two different culture media. Immature oocytes were isolated from ovaries collected from domestic gilts at a local slaughterhouse and were co-cultured with follicle shells for in vitro maturation (Hoshino et al. 2003 Theriogenology 59, 260). Donor cells were collected from fibroblast cells of miniature Potbelly pigs. Cells synchronized at the G1-phase were prepared shortly after dividing M-phase cells that had been synchronized using 2-methoxyestradiol, as described by Urakawa et al. (2004 Theriogenology 62, 714–728). The G0/G1 cells were also prepared from a fully confluent culture of cells. Donor cells were fused with enucleated oocytes and simultaneously activated by two electric pulses. Reconstructed embryos were cultured in two different media [Whitten and Biggers medium supplemented with 0.5 mg/mL hyaluronic acid sodium salt (WM) and porcine zygote medium-3 (PZM-3, Yoshioka et al. 2002 Biol. Reprod. 66, 112–119)] under 5% CO₂ in air. Cleavage rate and development rate to the blastocyst stage were assessed after 48 and 168 hr of culture, respectively. The results are summarized in Table 1. Developmental rate to the blastocyst stage of SCNT embryos reconstructed from G1 cells and cultured in PZM-3 (40%) was significantly higher than that of embryos cultured in WM (25%). The SCNT embryos of the G1 cells showed significantly lower cleavage rate (51%) than that of the G0/G1 cells (69%). However, the developmental rates to the blastocyst stage per cleaved embryo in WM were significantly higher in G1 cells (50%) compared with G0/G1 cells (32%). In addition, the total cell number of the SCNT blastocysts was comparable between the cultures in WM (58 ± 4) and PZM-3 (46 ± 5), although the ratio of inner cell mass cells to the total cell number was significantly higher in PZM-3 (32%) compared with WM (14%). These results suggest that PZM-3 may fit with the culture of SCNT embryos, and that the G1 synchronized cells could be stably reprogrammed for early embryonic development in SCNT embryos and be useful as donor cells for analyzing the processes of nuclear reprogramming.

Table 1.
Development of nuclear transfer embryos reconstructed from G1 or G0/G1 cells in different culture media