Parthenogenetic development of porcine oocytes treated by ethanol, cycloheximide, cytochalasin B and 6-dimethylaminopurine.
This study was carried out to investigate the various concentrations and exposure times of ethanol, one of many intracellular calcium elevating agents, and a sequential combination of ethanol (8%), cycloheximide (CHX, 10 microg/ml), cytochalasin B (CCB, 7.5 microg/ml) and 6-dimethylaminopurine (6-DMAP, 2 mM) to improve parthenogenetic activation and development of in vitro matured porcine oocytes. Cumulus-oocyte complexes (COCs) were matured in tissue culture medium (TCM) 199 for 44 h at 38.5 degrees C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were activated by concentrations of 0, 5, 6, 7, 8, 9 and 10% ethanol for 10 min and exposure times of 0, 5, 8, 10, 12 and 15 min with 8% ethanol in HEPES buffered (25 mM) NCSU-23 medium. Also, oocytes were activated with the NCSU-23 medium containing 8% ethanol for 10 min. After that, oocytes were incubated in the NCSU-23 medium supplemented with CHX, CCB, 6-DMAP, CHX + CCB, CHX + 6-DMAP, CCB + 6-DMAP and CHX + CCB + 6-DMAP for 3h, respectively. Following activation, oocytes were transferred into the NCSU-23 medium containing 0.4% BSA for further culture of 20 and 144 h at 38.5 degrees C, 5% CO2 in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly, more oocytes (29.3-33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8-15 min. Oocytes treated by chemical agents (40.5-70.5%) after exposure to ethanol significantly improved the rate of oocyte activation compared with ethanol alone (31.2%). The percentage of cleaved oocytes was higher in the ethanol+CHX+CCB+6-DMAP treatment (66.4%) than in other treatments (24.9-57.6%). Also, the rate of blastocyst formation was higher in the ethanol+CHX+CCB+6-DMAP treatment (25.0%) than in other treatments (0.0-19.3%). In conclusion, the optimal activation treatment of ethanol exposure alone for the in vitro matured porcine oocytes was 8% ethanol for 8-15 min. Oocytes activated by 8% ethanol for 10 min and incubated in the NCSU-23 medium supplemented with CHX, CCB and 6-DMAP for 3 h were more efficient for parthenogenetic development of in vitro matured porcine oocytes.
Yi
Y J
YJ
Division of Animal Science and Resources, Research Center for Transgenic Cloned Pigs, Chungnam National University, 305-764 Daejeon, Korea.
Park
C S
CS
D000225:Adenine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002970:Cleavage Stage, Ovum / Q000502:physiology; D003513:Cycloheximide / Q000008:administration & dosage / Q000494:pharmacology*; D003571:Cytochalasin B / Q000008:administration & dosage / Q000494:pharmacology*; D000431:Ethanol / Q000008:administration & dosage / Q000494:pharmacology*; D005260:Female; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology; D010312:Parthenogenesis / Q000187:drug effects*; D013552:Swine
D003571:Cytochalasin B; D000431:Ethanol; C001020:N(6),N(6)-dimethyladenine; D003513:Cycloheximide; D000225:Adenine