Liu L, Ju J C, Yang X
Department of Animal Science, University of Connecticut, Storrs.
Mol Reprod Dev. 1998 Mar;49(3):298-307. doi: 10.1002/(SICI)1098-2795(199803)49:3<298::AID-MRD10>3.0.CO;2-T.
Development of an effective activation protocol is of great importance for studying oocyte competence and embryo cloning. Experiments were designed to examine effects of intracellular calcium elevating agents such as calcium ionophore A23187 (CaA) and ethanol, or protein synthesis and phosphorylation inhibitors such as cycloheximide (CH) and 6-dimethylaminopurine (6-DMAP), or a sequential combination of these agents on both parthenogenetic development and protein patterns of newly matured bovine oocytes. Oocytes were matured for 24 hr in M-199 supplemented with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol at 39 degrees C in humidified air. They were then activated by various treatments and cultured in KSOM. Protein patterns at 15 hr after treatment were determined on 8-15% gradient SDS-PAGE and silver stained. Results demonstrated that none of the chemical agents--CaA, ethanol, 6-DMAP, or cycloheximide--could effectively induce parthenogenetic development of young bovine oocytes. When compared with the single treatments, sequentially combined treatments of CaA with 6-DMAP or with cycloheximide plus cytochalasin D (CD) significantly increased the rates of cleavage (78-82% versus 3-13%) and blastocyst development (31-40% versus 0%), which were comparable with those of IVF group (80% and 35%, respectively; P > 0.05). Supplementation with CD to the combined CaA and CH treatment improved rates of cleavage and blastocyst development versus without CD supplementation (31% versus 7%; P < 0.05). Fluorescent microscopy revealed that 95% (n = 40) of oocytes treated with CaA plus 6-DMAP had one pronucleus (PN) and one polar body (PB), while 88% (n = 40) in the CaA plus cycloheximide-treated group had one PN and two PBs and 85% (n = 40) in CaA plus cycloheximide and CD group had two PNs and one PB. Treatment by CaA alone resulted in 73% of oocytes (n = 40) arrested at a metaphase stage with two PBs (named as metaphase III or MIII). Protein patterns were similar for chemically activated and in vitro-fertilized (IVF) oocytes in that the 138- and 133-kDa proteins, whose functions are not yet known, were present in the metaphase-stage (MII 24 hr, MII 40 hr, and MIII) oocytes but were absent in PN-stage oocytes regardless of treatment. Therefore, these proteins seem to be metaphase-associated proteins. Taken together, we conclude that optimal parthenogenetic development of newly matured bovine oocytes can be obtained by calcium ionophore treatment followed by incubation in either 6-DMAP or cycloheximide plus cytochalasin D and that the reduction of the 138- and 133-kDa proteins might be necessary for the full activation of bovine oocytes.
开发一种有效的激活方案对于研究卵母细胞的能力和胚胎克隆至关重要。实验旨在研究细胞内钙升高剂（如钙离子载体A23187（CaA）和乙醇）、蛋白质合成和磷酸化抑制剂（如放线菌酮（CH）和6 - 二甲基氨基嘌呤（6 - DMAP））或这些试剂的顺序组合对新成熟牛卵母细胞孤雌发育和蛋白质模式的影响。卵母细胞在补充有促卵泡激素（FSH）、促黄体生成素（LH）和雌二醇的M - 199中于39℃、湿润空气中成熟24小时。然后通过各种处理对其进行激活，并在KSOM中培养。处理后15小时的蛋白质模式通过8 - 15%梯度SDS - PAGE测定并进行银染。结果表明，化学试剂CaA、乙醇、6 - DMAP或放线菌酮均不能有效诱导年轻牛卵母细胞的孤雌发育。与单一处理相比，CaA与6 - DMAP或与放线菌酮加细胞松弛素D（CD）的顺序联合处理显著提高了卵裂率（78 - 82%对3 - 13%）和囊胚发育率（31 - 40%对0%），这与体外受精组相当（分别为80%和35%；P>0.05）。与不添加CD的CaA和CH联合处理相比，添加CD可提高卵裂率和囊胚发育率（31%对7%；P<0.05）。荧光显微镜检查显示，用CaA加6 - DMAP处理的卵母细胞中有95%（n = 40）有一个原核（PN）和一个极体（PB），而CaA加放线菌酮处理组中有88%（n = 40）有一个PN和两个PB，CaA加放线菌酮和CD组中有85%（n = 40）有两个PN和一个PB。单独用CaA处理导致73%的卵母细胞（n = 40）停滞在中期阶段，有两个PB（称为中期III或MIII）。化学激活和体外受精（IVF）的卵母细胞的蛋白质模式相似，即功能尚不清楚的138 kDa和133 kDa蛋白质存在于中期阶段（MII 24小时、MII 40小时和MIII）的卵母细胞中，但在PN阶段的卵母细胞中不存在，无论处理方式如何。因此，这些蛋白质似乎是与中期相关的蛋白质。综上所述，我们得出结论，新成熟牛卵母细胞的最佳孤雌发育可通过钙离子载体处理，然后在6 - DMAP或放线菌酮加细胞松弛素D中孵育获得，并且138 kDa和133 kDa蛋白质的减少可能是牛卵母细胞完全激活所必需的。
