Parthenogenetic activation of porcine oocytes and isolation of embryonic stem cells-like derived from parthenogenetic blastocysts.

摘要

The study was conducted to optimize the electrical activation parameters, parthenogenetic activation methods, in vitro embryo culture and isolation of embryonic stem cells-like (ESCs) derived from porcine parthenogenetic blastocysts (pPBs). Results revealed that the electric field strength increased from 1.0 to 2.7 kV/cm, and the cleavage rate of parthenogenetic embryos increased gradually but the rate of oocyte lysis was significantly increased when using 2.7 kV/cm field strength. The rate of cleavage in 2.2 and 2.7 kV/cm groups was significantly increased in comparison with 1.0 kV/cm group. A voltage field strength of 2.2 kV/cm DC was used to investigate blastocyst development following activation with a single pulse of 30 or 60- micro sec pulse duration. The observed optimum pulse duration was 30- micro sec with a blastocyst rate of 20.7%. Multiple pulses were inferior to a single pulse for blastocyst yield (8.0 vs. 29.9%) (p<0.05). For porcine oocyte parthenogenetic activation methods, the rates of cleavage (79.0 vs. 59.8%) and blastocysts (19.4 vs. 3.4%) were significantly increased in electrical activation in contrast to chemical activation with ionomycin/6-DMAP (p<0.05). Rates of cleavage and blastocyst formation in NCSU-23 and PZM-3 embryo media were higher than G1.3/G2.3 serial culture media, but there was no significant difference among the three groups. The total cell number of blastocysts in PZM-3 embryo culture media containing 5 micro g/ml insulin was significantly higher than the control (44.3+or-.1 vs. 33.9+or-1.7). For isolation of PESCs-like, the rates of porcine blastocysts attached to feeder layers and ICM colony formation in Method B (nude embryo culture) were better than those in Method A (intact embryo culture).