Effects of duration, concentration, and timing of ionomycin and 6-dimethylaminopurine (6-DMAP) treatment on activation of goat oocytes.
The protocol of ionomycin followed by 6-dimethylaminopurine (6-DMAP) is commonly used for activation of oocytes and reconstituted embryos. Since numerous abnormalities and impaired development were observed when oocytes were activated with 6-DMAP, this protocol needs optimization. Effects of concentration and treatment duration of both drugs on activation and development of goat oocytes were examined in this study. The best oocyte activation (87-95%), assessed by pronuclear formation, was obtained when oocytes matured in vitro for 27 hr were treated with 0.625-20 microM ionomycin for 1 min before 6-hr incubation in 2 mM 6-DMAP. Progressional reduction of time for 6-DMAP-exposure showed that the duration of 6-DMAP treatment can be reduced to 1 hr from the second up to the fourth hour after ionomycin, to produce activation rates greater than 85%. Activation rates of oocytes in vitro matured for 27, 30, and 33 hr were higher (P < 0.05) than that of oocytes matured for 24 hr when treated with ionomycin plus 1-hr (the third hour) 6-DMAP, but a 4-hr incubation in 6-DMAP enhanced activation of the 24-hr oocytes. Goat activated oocytes began pronuclear formation at 3 hr and completed it by 5-hr post ionomycin. An extended incubation in 6-DMAP (a) impaired the development of goat parthenotes, (b) quickened both the release from metaphase arrest and the pronuclear formation, and (c) inhibited the chromosome movement at anaphase II (A-II) and telophase II (T-II), leading to the formation of one pronucleus without extrusion of PB2. In conclusion, duration, concentration, and timing of ionomycin and 6-DMAP treatment had marked effects on goat oocyte activation, and to obtain better activation and development, goat oocytes matured in vitro for 27 hr should be activated by 1 min exposure to 2.5 microM ionomycin followed by 2 mM 6-DMAP treatment for the third hour.
Lan
Guo-Cheng
GC
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China.
Han
Dong
D
Wu
Yan-Guanng
YG
Han
Zheng-Bin
ZB
Ma
Suo-Feng
SF
Liu
Xin-Yong
XY
Chang
Chong-Le
CL
Tan
Jing-He
JH
D000225:Adenine / Q000031:analogs & derivatives*; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D006041:Goats; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology*; D008540:Meiosis / Q000187:drug effects*; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*
D007476:Ionophores; D047428:Protein Kinase Inhibitors; D015759:Ionomycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine