OOLEMMAL PROTEOMICS

这项资助计划的目标是鉴定新的卵细胞质膜(卵膜)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。我们实验室最近利用二维(2-D)聚丙烯酰胺凝胶电泳法开始建立小鼠卵母细胞蛋白质组数据库(OPD)，迄今已有500多个银染蛋白质被解析。用生物素标记细胞表面已经确定了大约50种可能的鸡蛋表面蛋白的子集，即鸡蛋表面指数(ESI)。来自几个表面标记蛋白质的微序列数据表明，有一个蛋白质是新的。另一种表面标记的蛋白质是钙网蛋白，在体外表明细胞表面的钙网蛋白是受精所必需的。此外，两个表面标记的蛋白质是PI-PLC敏感的，在体外表明这些蛋白质是精子-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数据库并鉴定至少五个新的表面标记蛋白质来进一步发展我们的初步进展。这些蛋白质将被克隆、鉴定和表达，并将研究它们在精子-卵子相互作用中的功能作用。然后将在小鼠身上进行生育试验，以测试卵母细胞特有的新型卵膜蛋白的免疫避孕潜力。这项赠款提案的具体目标是：1)创建一个全面的小鼠卵母细胞蛋白质组数据库(OPD)，并确定表面标记蛋白质的子集，即鸡蛋表面指数(ESI)。2)利用串联质谱仪获得卵膜蛋白的氨基酸微序列数据。3)克隆和测序代表至少5个新的卵膜蛋白完整开放阅读框架的cDNA。4)探讨新的卵膜蛋白的功能作用。5)在小鼠生育试验中评价卵膜蛋白的免疫避孕作用。这一研究结果将促进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.