Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro.
The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
Eiji
E
Mizutani
Jin-Yi
JY
Jiang
Satoshi
S
Mizuno
Ikuo
I
Tomioka
Tadahiro
T
Shinozawa
Jin
J
Kobayashi
Hiroshi
H
Sasada
Eimei
E
Sato
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001757:Blastomeres / Q000502:physiology; D003470:Culture Media; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro; D066298:In Vitro Techniques; D009865:Oocytes / Q000502:physiology*; D009994:Osmolar Concentration; D010312:Parthenogenesis / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar
D003470:Culture Media; D004791:Enzyme Inhibitors; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine