Liang Y Y, Ye D N, Laowtammathron C, Phermthai T, Nagai T, Somfai T, Parnpai R
Embryo Technology and Stem Cell Research Center and School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Reprod Domest Anim. 2011 Feb;46(1):e67-73. doi: 10.1111/j.1439-0531.2010.01636.x.
The objective of this study was to optimize the activation protocol for buffalo oocytes after intracytoplasmic sperm injection (ICSI). The release of the second polar body (PB) at 3, 6 and 9 h after ICSI of in-vitro matured oocytes activated either with 5 μm ionomycin (Io) or with 7% ethanol (EtOH) was preliminary examined. The highest rate of second PB extrusion occurred at 3 h of activation, and the second PB extrusion in EtOH group was significantly higher than that in Io group. Oocytes that extruded the second PB were selected and cultured either with 1.9 mm 6-dimethylaminopurine (6-DMAP) for 3 h or with 10 μg/ml cycloheximide (CHX) for 5 h. Significantly higher rate of oocytes formed 2 pronuclei in EtOH combined with CHX (EtOH + CHX) (62%) group compared to those of Io + CHX (42%) and EtOH + 6-DMAP (48%) groups (p<0.01) whereas Io + 6-DMAP group showed intermediate value (58%). Significantly higher blastocyst formation rates were obtained in Io + 6-DMAP (29%) and EtOH + CHX (24%) groups than in Io + CHX (6%) and EtOH + 6-DMAP (17%) groups. Our results indicate that buffalo ICSI oocytes are effectively activated by combination treatment of Io with 6-DMAP and EtOH with CHX resulting in the highest cleavage and blastocyst formation rates.
本研究的目的是优化水牛卵母细胞胞浆内单精子注射（ICSI）后的激活方案。初步检测了用5μm离子霉素（Io）或7%乙醇（EtOH）激活体外成熟卵母细胞后，在ICSI后3、6和9小时第二极体（PB）的释放情况。第二PB挤出率最高出现在激活后3小时，且EtOH组的第二PB挤出率显著高于Io组。选择挤出第二PB的卵母细胞，用1.9mM 6-二甲基氨基嘌呤（6-DMAP）培养3小时或用10μg/ml环己酰亚胺（CHX）培养5小时。与Io + CHX（42%）组和EtOH + 6-DMAP（48%）组相比，EtOH联合CHX（EtOH + CHX）组（62%）形成双原核的卵母细胞率显著更高（p<0.01），而Io + 6-DMAP组显示出中间值（58%）。Io + 6-DMAP组（29%）和EtOH + CHX组（24%）的囊胚形成率显著高于Io + CHX组（6%）和EtOH + 6-DMAP组（17%）。我们的结果表明，Io与6-DMAP联合处理以及EtOH与CHX联合处理能有效激活水牛ICSI卵母细胞，从而获得最高的卵裂率和囊胚形成率。
