Wang W, Sun Q, Hosoe M, Shioya Y
National Institute of Animal Industry, Tsukuba, Japan.
Zygote. 1997 Feb;5(1):75-82. doi: 10.1017/s0967199400003580.
The dependence of pig oocyte activation (both nuclear activation and cortical granule exocytosis) induced by staurosporine on intracellular Ca2+ rise and spindle assembly was studied. Nuclear activation was evaluated by pronuclear (PN) formation, cleavage and their developmental ability, and cortical granule (CG) exocytosis was assessed by electron microscopy and laser confocal microscopy of oocytes labelled with fluorescein isothiocyanate-peanut agglutinin. Exposure of pig oocytes of 0.3 and 3 microM protein kinase inhibitor staurosporine for 30 min resulted in the nuclear activation in 71.8% and 85.7% of the oocytes, respectively. The pronuclei in activated oocytes contained several compact nucleoli. When the cleaved 2-cell oocytes were further cultured in vitro, 93.5% developed beyond the 4-cell stage, and 12.9% developed to the morula stage after 4 days of culture. Of the oocytes treated with 3 microM staurosporine, 62.5% and 9.4% released their CGs partially and completely, respectively. The nuclear activation induced by staurosporine was overcome by the prior treatment of oocytes with okadaic acid, resulting in only 33.3% of the oocytes undergoing nuclear activation. However, when oocytes were exposed first to 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethanal ester), a cell permeate calcium chelator, or Colcemid, a meiotic spindle disrupter, and then to staurosporine, nuclear activation was observed in 74.2% and 82.3% of the oocytes, respectively. These data were the same as those in oocytes treated only with staurosporine (85.7%). The present study indicates that pig oocytes can be activated by the inhibition of staurosporine-sensitive protein kinase(s), and that this activation is dependent upon mitogen-activated protein kinase but independent of the intracellular Ca2+ rise and spindle integrity.
研究了星形孢菌素诱导的猪卵母细胞激活（包括核激活和皮质颗粒胞吐）对细胞内钙离子升高和纺锤体组装的依赖性。通过原核（PN）形成、卵裂及其发育能力评估核激活，通过电子显微镜和异硫氰酸荧光素 - 花生凝集素标记的卵母细胞激光共聚焦显微镜评估皮质颗粒（CG）胞吐。将猪卵母细胞暴露于0.3微摩尔和3微摩尔的蛋白激酶抑制剂星形孢菌素中30分钟，分别导致71.8%和85.7%的卵母细胞核激活。激活的卵母细胞中的原核含有几个紧密的核仁。当卵裂的2 - 细胞卵母细胞进一步体外培养时，93.5%发育到4 - 细胞期以上，培养4天后12.9%发育到桑椹胚期。用3微摩尔星形孢菌素处理的卵母细胞中，分别有62.5%和9.4%部分和完全释放其皮质颗粒。用冈田酸预先处理卵母细胞可克服星形孢菌素诱导的核激活，导致仅33.3%的卵母细胞发生核激活。然而，当卵母细胞首先暴露于细胞可渗透的钙螯合剂1,2 - 双（O - 氨基苯氧基）乙烷 - N，N，N'，N' - 四乙酸（乙酰氧甲酯）或减数分裂纺锤体破坏剂秋水仙酰胺，然后再暴露于星形孢菌素时，分别有74.2%和82.3%的卵母细胞核激活。这些数据与仅用星形孢菌素处理的卵母细胞（85.7%）的数据相同。本研究表明，猪卵母细胞可通过抑制对星形孢菌素敏感的蛋白激酶而被激活，并且这种激活依赖于丝裂原活化蛋白激酶，但与细胞内钙离子升高和纺锤体完整性无关。
