Proteasomal interference prevents zona pellucida penetration and fertilization in mammals
Proteasomal interference prevents zona pellucida penetration and fertilization in mammals
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DOI：
10.1095/biolreprod.104.032532
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发表时间：
2004-11-01
影响因子：
3.6
通讯作者：
Day, BN
中科院分区：
文献类型：
--
作者：
Sutovsky, P;Manandhar, G;Day, BN
泛素-蛋白酶体途径与海鞘精子穿透海鞘卵黄包膜有关（Sawada et al.，Proc Natl Acad Sci U S A 2002; 99：1223-1228）。本研究提供了实验证据，证明蛋白酶体参与哺乳动物透明带（ZP）的渗透。以猪体外受精为模型，特异性蛋白酶体抑制剂MG-132和lactacystin可完全抑制ZP的穿透。识别20 S蛋白酶体核心亚基β-1 i、β-2 i、α-6和β-5的三种商业兔血清在非常低的浓度下（即，在受精培养基中稀释1/2000至1/8000）。无论是蛋白酶体抑制剂，也没有抗体有任何影响精子ZP结合和顶体胞吐在透明带封闭的卵母细胞或受精率在透明带的卵母细胞，这是高度多精子。与泛素-蛋白酶体途径在ZP穿透中的可能作用一致，通过特异性抗体检测到猪精子顶体中的泛素和各种α和β型蛋白酶体亚基，免疫沉淀和MALDI-TOF微测序从猪精子提取物。通过落射荧光显微镜在ZP的外表面上检测到抗泛素免疫反应底物。因此，这项研究提供了有力的证据，暗示泛素-蛋白酶体途径在哺乳动物受精和受精卵穿透。这一发现开辟了一条新的顶体/ZP研究路线，因为对精子顶体蛋白酶体的进一步研究可以为体外受精过程中多精子症的管理提供新的工具，并确定避孕开发的新靶点。
The ubiquitin-proteasome pathway has been implicated in the penetration of ascidian vitelline envelope by the fertilizing spermatozoon (Sawada et al., Proc Natl Acad Sci U S A 2002; 99:1223-1228). The present study provides experimental evidence demonstrating proteasome involvement in the penetration of mammalian zona pellucida (ZP). Using porcine in vitro fertilization as a model, penetration of ZP was completely inhibited by specific proteasomal inhibitors MG-132 and lactacystin. Three commercial rabbit sera recognizing 20S proteasomal core subunits beta-1i, beta-2i, alpha-6, and beta-5 completely blocked fertilization at a very low concentration (i.e., diluted 1/2000 to 1/8000 in fertilization medium). Neither proteasome inhibitors nor antibodies had any effects on sperm-ZP binding and acrosome exocytosis in zona-enclosed oocytes or on fertilization rates in zona-free oocytes, which were highly polyspermic. Consistent with a possible role of ubiquitin-proteasome pathway in ZP penetration, ubiquitin and various alpha and beta type proteasomal subunits were detected in boar sperm acrosome by specific antibodies, immunoprecipitated and microsequenced by MALDI-TOF from boar sperm extracts. Antiubiquitin-immunoreactive substrates were detected on the outer face of ZP by epifluorescence microscopy. This study therefore provides strong evidence implicating the ubiquitin-proteasome pathway in mammalian fertilization and zona penetration. This finding opens a new line of acrosome/ZP research because further studies of the sperm acrosomal proteasome can provide new tools for the management of polyspermia during in vitro fertilization and identify new targets for contraceptive development.