Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer
Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer
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DOI：
10.1017/s0967199405003370
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发表时间：
2005-11-01
期刊：
影响因子：
1.7
通讯作者：
Kikuchi, K
中科院分区：
文献类型：
--
作者：
Iwamoto, M;Onishi, A;Kikuchi, K
研究了用咖啡因处理过的猪卵母细胞来抑制m期促进因子活性下降的可能性。卵丘-卵母细胞复合物（COCs）初始培养36小时，随后加或不加5 mM咖啡因培养24小时（60CA+组和60CA-组共培养60小时）。作为对照组，COCs在不含咖啡因（48CA-）的情况下培养48 h。与48CA-组相比，60CA+组和60CA-组电刺激后10 h原核形成率显著降低（p < 0.05）。60CA-组的骨裂率显著高于60CA+和48CA-组（p < 0.05）。刺激卵母细胞培养6 d后，60CA+组囊胚形成率显著低于48CA-组，破碎或变性率显著高于48CA-组（p < 0.05）。然而，囊胚总细胞数量不受成熟时间和咖啡因处理的影响。在未去核卵母细胞成熟完成前（48 h或60 h），将体细胞核注入卵母细胞并与细胞质接触一定时间（1 ~ 11 h）， 60CA-卵母细胞与细胞质接触1 ~ 2 h后核膜破裂率显著低于其他实验组（p < 0.05）。同一60CA-组的分散染色体形成率比其他组低（p = 0.08）。在去核和转移细胞核后，60CA+和60CA-组囊胚形成率显著低于48CA-组（p < 0.05）。各组胚泡质量无显著差异。这些结果表明，移植体细胞核的染色体去浓缩受到细胞质暴露时间和受体猪卵母细胞年龄的影响，咖啡因处理促进核重塑，但不能阻止卵母细胞老化引起的克隆胚胎发育能力下降。
The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus-oocyte complexes (COCs) were cultured initially for 36 h and subsequently with or without 5 mM caffeine for 24 h (in total for 60 h: 60CA+ or 60CA- group, respectively). As a control group, COCs were cultured for 48 h without caffeine (48CA-). The pronuclear formation rates at 10 h after electrical stimulation in the 60CA+ and 60CA- groups decreased significantly (p < 0.05) compared with the 48CA- group. However, the fragmentation rate was significantly higher (p < 0.05) in the 60CA- group than in the 60CA+ and 48CA- groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p < 0.05) than the 48CA- group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1-11 h) before the completion of maturation (48 or 60 h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1-2 h in the 60CA- oocytes was significantly lower (p < 0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA- group tended to be lower (p = 0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA- groups were significantly lower (p < 0.05) than in the 48CA- group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.