Studies in a number of mammals indicate that 1-5% of fertilized eggs develop abnormally into triploid embryonic abortuses. In the human this is primarily due to fertilization by more than one sperm. The egg's primary means of preventing polyspermy is the fertilization-associated release of cortical granules (CGs) whose contents biochemically modify the egg's zona pellucida making it less penetrable to sperm. In antral follicle oocytes (pre-metaphase II) which have not completed cytoplasmic maturation, one major cause of polyspermy appears to be their incompetence to undergo CG release. Preliminary evidence indicates that this incompetence involves the activation mechanism which includes intracellular calcium (Ca) and protein kinase C (PKC). The long-range goals of this proposal are to determine the causes for this incompetence in terms of Ca and PKC regulation as well as more accurately assess when competence develops in the mouse oocyte. The following are specific aims: 1. To determine when the oocyte develops the ability to undergo sperm- induced CG release. At appropriate stages of meiotic maturation, sperm- induced CG release will be assayed by % CG loss [quantitative image analysis(QIA)], by biochemical analysis of the zona (protein ZP2), and by % monospermic (vs polyspermic) fertilization. 2. To analyse the oocyte's competence to store and respond to intracellular Ca during meiotic maturation. The development of cellular components involved in calcium regulation will be analyzed with specific fluorescent probes and QIA. The ability of the developing oocyte to undergo CG loss in response to (microinjected) intracellular calcium will be assessed. 3. To analyse the oocyte's competence to undergo CG release in response to protein kinase C activators during meiotic maturation. At appropriate stages of maturation, the ability of PKC agonists to induce CG release will be investigated by QIA and ZP2 analysis. 4. To test for the synergism of Ca and PKC agonists in activating the pathways leading to CG loss. Synergism will be investigated in mature eggs and, if found, its development will be traced during meiotic maturation. These studies should result in 1) a more detailed explanation of the mechanism of egg and CG activation, 2) an identification of the stages of oocyte maturation which are most susceptable to polyspermic fertilization, 3) at least one likely scientific explanation for the origin of abnormal triploid abortuses observed in some mammalian species.
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