The suppression of fragmentation by stabilization of actin filament in porcine enucleated oocytes.
A thorough understanding of the mechanism underlying fragmentation would contribute to the improvement of the developmental ability of reconstructed embryos after nuclear transfer. We conducted the present study to elucidate the influence of the nuclear transfer method on fragmentation of enucleated oocytes and the relationship between change in actin filament distribution and fragmentation. In Experiment 1, we examined activation rates of in vitro matured oocytes. These were 12.9% in maturation alone, 75.7% in electrical stimulation, and 57.9% in ethanol/cycloheximide treatment. In Experiment 2, we observed a higher rate of fragmentation (P < 0.05) in cultured oocytes that had been enucleated and electrically stimulated than in oocytes subjected to the other treatments (maturation alone, enucleation alone and enucleation plus ethanol/cycloheximide activation). In Experiment 3, we stained enucleated and electrically stimulated oocytes with rhodamine/phalloidin dye to show discontinuous distributions in the ooplasm of treated oocytes; oocytes in the other treatment groups showed homogenous distributions of actin filaments (AFs). In Experiment 4, we added cytochalasin B, an inhibitor of AF polymerization, to the culture medium, which prevented fragmentation of enucleated plus electrically stimulated oocytes (cytochalasin B, [+] 0.0%, [-] 60.7% at 24 h after treatment, P < 0.05). In Experiment 5, we investigated the relationship between fragmentation and alteration in AF distribution in enucleated plus electrically stimulated oocytes. At 0 h of culture, enucleated plus electrically stimulated oocytes showed discontinuous distributions of AFs, while nontreated oocytes showed homogenous AF distributions. At 24 and 48 h of culture, fragmentation proceeded in enucleated plus electrically stimulated oocytes and the discontinuous AF distribution diminished with time. In Experiment 6, we added hyaluronic acid (HA) to the culture medium, which suppressed fragmentation of enucleated plus electrically stimulated oocytes (HA, [+] 28.5%, [-] 66.4% at 24 h after treatment, P < 0.05). The results suggest that electrical stimulation induces a change in the AF distribution of oocytes, resulting in fragmentation, and that the addition of HA to the culture media is effective for the suppression of fragmentation.
Kawahara
Manabu
M
Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University, Sapporo-shi, Japan.
Mori
Tadashi
T
Tanaka
Hozumi
H
Shimizu
Hiroshi
H
D000199:Actins / Q000032:analysis / Q000737:chemistry / Q000648:ultrastructure*; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry / Q000648:ultrastructure*; D004558:Electric Stimulation; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes; D006820:Hyaluronic Acid / Q000494:pharmacology; D018613:Microscopy, Confocal; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D010590:Phalloidine; D012235:Rhodamines; D013552:Swine
D000199:Actins; D005456:Fluorescent Dyes; D012235:Rhodamines; D010590:Phalloidine; D000431:Ethanol; D006820:Hyaluronic Acid; D003513:Cycloheximide