Differential effects of protein synthesis inhibitors on porcine oocyte activation
Differential effects of protein synthesis inhibitors on porcine oocyte activation
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蛋白质合成抑制剂对猪卵母细胞活化的不同影响
DOI：
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发表时间：
1995
影响因子：
2.5
通讯作者：
R. Prather
中科院分区：
文献类型：
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作者：
Diana J. Nussbaum;R. Prather
本研究旨在探讨放线菌酮和嘌呤霉素对猪卵母细胞激活和蛋白质合成的影响。成熟卵母细胞经电刺激后，在放线菌酮（5 μ/ml）存在下培养6或24小时，92%的卵母细胞被激活，原核形成率为2.8%;未电刺激但用放线菌酮培养的卵母细胞激活率为5.3%;当在存在放线菌酮、嘌呤霉素（100 μg/ml）或不存在蛋白质合成抑制剂的情况下与L-[35 S]蛋氨酸培养24 h时，卵母细胞的平均放射性标记掺入率分别为36.5、2.21和32.0 fmol/4 h/卵母细胞。因此，放线菌酮在培养24小时后对蛋白质合成几乎没有影响。1D-SDS PAGE显示，用嘌呤霉素或放线菌酮培养的卵母细胞未被激活，而电刺激的卵母细胞被激活，其特征在于25-kDa多肽转化为22-kDa多肽。重复放射性标记实验，除了卵母细胞培养4或24小时。在4小时，放线菌酮组的平均掺入率较低（2.34 fmol/4小时/卵母细胞），但嘌呤霉素组（15.7 fmol/4小时/卵母细胞）和对照组（18.9 fmol/4小时/卵母细胞）相似。在24小时，嘌呤霉素组（5.73 fmol/4 hr/卵母细胞）的掺入率较低，而放线菌酮组（22.6 fmol/4 hr/卵母细胞）和对照组（26.0 fmol/4 hr/卵母细胞）相似。放线菌酮在培养过程中更早更有效，而嘌呤霉素更晚更有效。当与ES联合使用时，嘌呤霉素的激活率（87.8%）确实高于（P = 0.10）单独电刺激（73.0%）。最后一个实验评估了转移到结扎的输卵管后胚泡的发育。环己酰亚胺处理结合电脉冲并没有增加致密桑葚胚或囊胚形成的速率。总之，嘌呤霉素和放线菌酮对蛋白质合成有不同的影响，虽然放线菌酮单独不会诱导猪卵母细胞的激活，但它与电刺激相结合在产生激活的卵母细胞方面非常有效。© 1995 Wiley利斯公司
This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L‐[35S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D‐SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25‐kDa polypeptide to a 22‐kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley‐Liss, Inc.