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.
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