78 EFFECTS OF DIFFERENT ACTIVATION PROTOCOLS ON ACTIN FILAMENT DISTRIBUTION AND IN VITRO DEVELOPMENT OF MINIATURE PIG NT EMBRYOS

Abstract

In the process of producing reconstructed oocytes nuclear transfer (NT) embryos by somatic cell nuclear transfer, in vitro-matured oocytes can be used as recipient ones. It, however, has been well documented that after IVF porcine embryos derived from in vitro-matured oocytes have a small number of cells and low viability compared from those in vivo. As one possible reason, abnormal actin filament distribution has been detected in abnormal embryo cleavage and small cell numbers (Wang et al. 1999 Biol. Reprod. 60, 1020-1028). Artificial activation, which is necessary for development of NT embryos, can affect actin filament distribution of porcine oocytes matured in vitro, resulting in fragmentation (Kawahara et al. 2002 Theriogenology 58, 1081-1095). In the present study, we investigated effects of different activation protocols on actin filament distribution and in vitro development of miniature pig NT embryos. Porcine oocytes collected from ovaries were matured in vitro for 40 to 44 h in NCSU-23. First, we compared different activation protocols in development rates to blastocysts of oocytes activated. We used three activation methods (15 �M ionomycin treatment for 20 min (I), double DC pulses of 1.2 kV/cm for 60 ms in intervals of 5 s (E), and 5 mg/mL cycloheximide treatment for 5 h (C)) to prepare seven activation protocols (I, E, C, I + C, I + E, E + C, and I + E + C). Second, we examined effects of different activation protocols on actin filament distribution and subsequent development of NT embryos activated by the different activation protocols. Matured oocytes were enucleated, and fused with miniature pig fetal fibroblasts in calcium-free medium; approximately 3 h later, the resultant NT embryos were activated with three activation protocols (E, I + C, or I + E + C). All data were analyzed by chi-square test. The developmental rates to blastocysts in the I, E, C, I + C, I + E, E + C, and I + E + C groups were 5.6, 11.1, 0.0, 36.1, 20.7, 14.6, and 24.7%, respectively, showing that the rate in oocytes activated with I + C was significantly higher (P < 0.05) than the rates in oocytes activated by other treatments. In NT embryos, the developmental rates to blastocysts in the E, I + C, or I + E + C groups were 4.1, 14.3, and 4.6%, respectively, showing that the rate in NT embryos activated with I + C was significantly higher (P < 0.05) than the rate in NT embryos activated with other treatments. The abnormal rate of actin filament distribution in NT embryos activated with E or I + E + C was significantly higher (P < 0.05) than that in NT embryos activated with I + C (26.7% or 33.3% vs. 6.7%). The present results suggest that in miniature pig NT embryos an activation protocol by ionomycin combined with cycloheximide treatments may avoid physical damage to actin filaments with the resultant improvement of subsequent development.