Murray G, Reed C, Marsden M, Rise M, Wang D, Burke R D
Department of Biology, University of Victoria, British Columbia, Canada.
Dev Biol. 2000 Nov 15;227(2):633-47. doi: 10.1006/dbio.2000.9910.
Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.
整合素表达于一些脊椎动物卵子的表面，据认为它们在受精过程中发挥作用。本研究的目的是确定整合素是否在海胆卵子上表达。使用同源聚合酶链反应克隆了αB和βC亚基。针对βC亚基和αB亚基细胞外结构域的细菌表达片段制备了单克隆抗体和多克隆抗体。此外，针对与βC细胞质结构域部分相对应的合成肽制备了单克隆抗体。用抗βC或抗αB免疫沉淀的生物素化卵皮质提取物分析产生了130 kDa和225 kDa的条带。免疫印迹证实βC是用抗αB免疫沉淀的复合物的一部分。共聚焦免疫荧光和免疫金电子显微镜显示，βC存在于未受精卵表面的微绒毛尖端和皮质颗粒中。在皮质反应期间，与βC和αB细胞外结构域抗体的免疫反应性从卵子表面消失，受精膜上的微绒毛铸型变得具有免疫反应性。用针对βC细胞质结构域的抗体，卵子表面的免疫反应性丧失，但受精膜不会立即变得具有免疫反应性。在卵皮质的免疫印迹中，有预测大小的αB和βC免疫反应条带。然而，在受精膜中，还存在一条分子量略低的第二条带。在大豆胰蛋白酶抑制剂存在下受精的卵子有伸长的微绒毛，这些微绒毛仍与升高的受精膜结合并对抗βC抗体有免疫反应性。在过氧化物酶抑制剂3-氨基-1,2,4-三唑存在下受精的卵子，其受精膜中βC免疫反应性呈斑片状分布。这些数据共同表明，αBβC整合素表达于未受精卵的表面，并且在皮质反应期间，细胞外结构域被蛋白酶切割并通过过氧化物酶交联到受精膜中。αBβC整合素受体在受精时被去除之前可能具有多种潜在功能，包括将卵黄膜附着到卵子表面以及锚定皮质细胞骨架。
