The overall goal of this proposal is to address the role of phospholipase C (PLC) isozymes in egg activation during fertilization, using the starfish egg as a model system. Briefly, isozymes will be identified by the reverse transcription polymerase chain reaction (RT PCR) using highly-conserved PLC oligos. Positive PCR products will then be used to screen an Asterina miniata oocyte cDNA library. Putative PLC clones will be sequenced and compared to known PLC sequences. The DNA sequence information will be used to design peptides for the production of polyclonal antibodies and for the synthesis of oligonucleotides. To assay for PLC function during fertilization, PLC antisense oligos or anti-PLC antibodies will be microinjected into the starfish egg in an effort to """"""""knock out"""""""" specific PLC isozymes. Several of the known responses of the egg to fertilization will be measured after oligo or antibody injection, including: the calcium wave, cortical granule exocytosis, the initiation of DNA synthesis, and cytokinesis. These studies are of interest because they will provide insight into the basic biology behind signal transduction during fertilization which may eventually lead to applications in contraception or the treatment of infertility.
