OOLEMMAL PROTEOMICS

这项拨款提案的目的是鉴定新的卵母细胞质膜（oolemma）蛋白，研究它们在精子-卵子相互作用中的作用，并测试它们的免疫避孕潜力。我们的实验室最近利用二维（2-D）聚丙烯酰胺凝胶电泳开始建立小鼠卵母细胞蛋白质组学数据库（OPD），迄今已解决了500多个银染色蛋白。用生物素对细胞表面进行标记已经鉴定出大约50种假定的卵表面蛋白，即卵表面指数（ESI）。来自几个表面标记蛋白的微序列数据表明，其中一个蛋白是新的。另一种表面标记蛋白是钙网蛋白，体外表明细胞表面钙网蛋白是受精所必需的。此外，两种表面标记的蛋白对PI-PLC敏感，体外表明这些蛋白是精子-卵子结合和融合所必需的。我们的目标是通过开发一个全面的小鼠卵母细胞蛋白质组学数据库并鉴定至少五种新的表面标记蛋白来建立我们的初步进展。这些蛋白将被克隆、表征和表达，并在精卵相互作用中的功能作用将被研究。生育试验将在小鼠中进行，以测试卵母细胞特异性新乳蛋白的免疫避孕潜力。该资助提案的具体目标是：1)创建一个全面的小鼠卵母细胞蛋白质组学数据库（OPD），并确定表面标记蛋白的子集，即卵表面指数（ESI）。2)利用串联质谱技术获得oolemma蛋白氨基酸微序列数据。3)克隆并测序至少5种新型乳蛋白的完整开放阅读框cdna。4)研究新型乳蛋白的功能作用。5)评价乳蛋白在小鼠生育试验中的免疫避孕潜力。本研究成果将推动受精生物学和免疫避孕领域的发展。

The objectives of this grant proposal are to identify novel oocyte plasma membrane (oolemma) proteins, investigate their role in sperm-egg interaction, and test their immunocontraceptive potential. Our laboratory has recently utilized two dimensional (2-D) polyacrylamide gel electrophoresis to begin building a mouse Oocyte Proteomic Database (OPD), with over 500 silver-stained proteins being resolved to date. Cell- surface labeling with biotin has identified a subset of approximately 50 putative egg surface proteins, the Egg Surface Index (ESI). Microsequence data from several of the surface-labeled proteins indicates that one protein is novel. Another surface labeled protein is caloreticulin and in vitro indicates that cell surface calreticulin is required for fertilization. Additionally, two surface-labeled proteins are PI-PLC sensitive and in vitro suggest that these proteins are required for sperm- egg binding and fusion. Our goal is to build on our initial progress by developing a comprehensive mouse oocyte proteomic database and identifying at least five novel surface labeled proteins. These proteins will be cloned, characterized, and expressed, and their functional roles in sperm-egg interaction will be investigated. Fertility trials will then be performed in mice to test the immunocontraceptive potential of oocyte- specific novel oolemmal proteins. The specific aims of this grant proposal are: 1) To create a comprehensive mouse Oocyte Proteomic Database(OPD) and identify a subset of surface-labeled proteins, the Egg Surface Index (ESI). 2) To obtain amino acid microsequence data from oolemma proteins using tandem mass spectroscopy. 3) To clone and sequences cDNAs representing the complete open reading frame of at least five novel oolemmal proteins. 4) To investigate the functional role of novel oolemmal proteins. 5) To evaluate the immunocontraceptive potential of oolemmal proteins in mouse fertility trials. Results from this research will advance the fields of fertilization biology and immunocontraception.