Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica)
Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica)
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DOI：
10.1530/rep-12-0165
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发表时间：
2012-10-01
期刊：
影响因子：
3.8
通讯作者：
Matsuda, Tsukasa
中科院分区：
文献类型：
--
作者：
Sasanami, Tomohiro;Sugiura, Kenichi;Matsuda, Tsukasa
在受精时，围绕禽类卵母细胞的细胞外基质，称为卵黄周膜（pvm），被精子传播的蛋白酶水解，尽管实际负责消化卵黄周膜的蛋白酶仍有待确定。在这里，我们展示了泛素-蛋白酶体系统在日本鹌鹑受精中起作用的证据。对纯化血清ZP1进行配体印迹分析，发现其诱导顶体反应和与ZP3结合的活性与pvm ZP1相似。用抗泛素抗体对纯化的ZP1和ZP3进行Western blot分析，发现只有pvm ZP1对抗体有反应。精子在pvm上的体外渗透实验表明，ZP1片段和完整的ZP3从pvm中释放出来。抗20s蛋白酶体抗体和超微结构分析表明，免疫反应性蛋白酶体定位于精子顶体区域。在孵育混合物中加入特异性蛋白酶体抑制剂MG132，或通过添加apyrase来消耗细胞外ATP，有效地抑制了pvm的精子穿孔。这些结果首次证明了精子蛋白酶体在鸟类受精过程中起重要作用，并且表明ZP1的胞外泛素化可能发生在其通过血液循环的运输过程中。复制（2012）144 423-431
At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin-proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody. In vitro penetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation. Reproduction (2012) 144 423-431