Effect of activation treatments on actin filament distribution and in vitro development of miniature pig somatic cell nuclear transfer embryos.
In the present study, we investigated the effect of activation treatments on the actin filament distribution and in vitro development of somatic cell nuclear transfer (SCNT) embryos in miniature pigs. We combined three activation methods, ionomycin (ION), electrical stimulation (ES), and cycloheximide treatment (CH), to prepare seven activation treatments (ION, ES, CH, ION + CH, ION + ES, ES + CH and ION + ES + CH). First, we investigated the activation rate of oocytes and in vitro development of parthenotes. The activation rates of the oocytes in the ION, ES, CH, ION + CH, ION + ES, ES + CH, and ION + ES + CH groups were 42.9, 51.3, 0.0, 82.1, 80.6, 78.1 and 78.6%, respectively, showing that the rates of the combined treatment groups were significantly higher (P<0.05) than those of the single treatment groups. Although there were no significant differences in the activation rates of the combined treatment groups, the developmental rate to blastocysts in the ION + CH treatment group (36.1%) was significantly higher (P<0.05) than the other combined treatment groups (14.6-24.7%). Subsequently, we investigated the in vitro development and distribution of microfilaments in SCNT embryos. The developmental rate to blastcysts of the SCNT embryos in the ION + CH treatment group (11.3%) was significantly higher (P<0.05) than in the ES and ION + ES + CH treatment groups (4.5 and 5.2%, respectively). The rate of normal actin filament distribution in the SCNT embryos activated with ION + CH was significantly higher (P<0.05) than those activated with ES or ION + ES + CH treatment (63.3 vs. 46.8 or 46.4%). In addition, the fragmentation rate of the SCNT embryos activated with ION + CH was significantly lower (P<0.05) than those activated with ION + ES + CH (14.9 vs. 26.1%). The present results suggest that an activation treatment of ionomycin combined with cycloheximide may avoid physical damage to microfilaments and result in improved subsequent development of miniature pig SCNT embryos.
Yamanaka
Ken-ichi
K
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University.
Sugimura
Satoshi
S
Wakai
Takuya
T
Shoji
Takehisa
T
Kobayashi
Jin
J
Sasada
Hiroshi
H
Sato
Eimei
E
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology*; D004558:Electric Stimulation; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology*; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D011247:Pregnancy; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D013552:Swine; D013556:Swine, Miniature
D007476:Ionophores; D011500:Protein Synthesis Inhibitors; D015759:Ionomycin; D003513:Cycloheximide