PARENT SESSION
REPRODUCTIVE TECHNOLOGIES

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(617) EFFECTS OF CELL CYCLE INHIBITORS ON SYNCHRONIZATION, PROLIFERATION, AND APOPTOSIS OF NEONATAL SKIN FIBROBLASTS IN RHESUS MONKEY.

Yang, Jifeng^{1, 2, 3}, Cai, Kejun^{1, 2, 3}, Ji, Weizhi^{1, 2, 3}, ¹ Department of Primate Biology and The Yunnan Key Laboratory of Animal Reproduction, Kunming, Yunnan, China² China-US Primate Biology Laboratory, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, Yunnan, China³ Graduate School of the Chinese Academy of Sciences, Beijing, Beijing, China

ABSTRACT- The qualities of oocytes and somatic cells and cell cycle coordination between them can primarily impose on the reprogramming process occurred in the reconstructed embryos. Treatments of donor cells before nuclear transfer make homogeneous cell population with high "clonability" available. To improve the synchronization of rhesus monkey fibroblasts, several methods including serum starvation, treatments with various cycle inhibitors (DMSO, roscovitine, aphidicoline and indirubin) were investigated in this study. Cell cycle synchronization was analyzed with a Becton Dickinson flow cytometer by synchronously measuring the DNA and protein contents. Compared with cycling cells (5.89%, 76.31%), serum starvation for 4 days with 0.5%FBS in MDEM significantly increased the proportions of G0 and G0+G1 cells to 37.19% and 96.62% respectively (p<0.05). Serum starvation for 6 days didn't further increase the synchronization effect. In the cycle inhibitors treatments, 1% (v/v) DMSO for 48 h can induce 32.54% G0 cells. 15M roscovitine for 24 h significantly increased (p<0.05) the proportion of G0+G1 cells (87.70%), but not G0 cells (6.84%) than that of cycling cells (79.32%, 7.05%). 6M aphidicolin for 14 h after serum starvation 2 days significantly increased (p<0.05) G0+G1 (96.57%) and G0 percentages (36.87%) compared with cycling cultures (79.32%, 7.05%). 10M indiruibin-3'-monoxime for 30 h increased (p<0.05) the proportions of G0+G1 (94.22%)and G0 cells (14.21%) compared with cycling cells (84.26%, 7.27%). By the DNA synthesis analysis with BrdU, it was shown that above arrest effect could be reversible when the treatments were removed. By TUNEL detection, it was shown that serum starvation induce more (p<0.05) apoptosis (5.81%, 5.45% and 5.60%) at 24, 48 and 96 h in monolayers besides floating cells than that in normal cultures (0.77%, 0.80% and 0.80%). However, there was no significant difference (p<0.05) on apoptosis in treatments with DMSO (0.90%), aphidicolin (1.99%), roscovitine (1.42%) or indirubin (1.82%) when compared with normal cycling cells (1.12%). These results showed the cycle inhibitors might be more effective and safe than serum starvation to synchronize the rhesus fibroblast for nuclear transfer.

KEY WORDS: cell cycle synchronization, cell cycle inhibitor, apoptosis, rhesus fibroblasts