In humans and other mammals, 1-5% of fertilized eggs become triploid, abort, and die. Human triploidy is primarily due to fertilization by more than one sperm and is usually prevented by the release of egg cortical granules (CGs) whose contents biochemically modify the egg's zona pellucida. In a mouse model, the long-term objectives are to identify:
(Aim 1) an important cause of triploidy that originates from a failure of CG release and (Aim 2) egg proteins responsible for the prevention of triploidy. The failure of CG release, associated with incomplete cytoplasmic maturation in mice and humans, involves second messengers and their targets: inositol trisphosphate (IP3), calcium (Ca), and Ca-dependent proteins.
Aims : 1) Development of Ca signaling for CG release: Does the ability to undergo CG exocytosis develop from increases in Ca release (hypothesis A) or an increased response to Ca (hypothesis B)? Prior to ovulation, is CG exocytosis failure due to insufficient elevation of Ca, a lack of sensitivity to Ca, and/or improper amount, localization, or phosphorylation of IP3 receptor isoforms (channels for intracellular Ca release)? 2) Biochemical characterization of individual (egg CG) proteins released at fertilization. Testing the hypothesis that one or more proteins are responsible for the zona protein (ZP3)-mediated block to polyspermy, characterized by reductions in sperm binding and in the acrosome reaction. Technical Methods: High resolution fluorescence microscopy and computerized image analysis of CGs, Ca imaging, electrophoresis, ultrasensitive chemiluminescence protein detection, and bioassays. Significance:
Aim 1) Identification of a signaling deficiency would provide an important 'marker' for oocyte cytoplasmic maturation and a more detailed scientific explanation for the origin of abnormal triploid abortuses. Determination of a quantitative relationship between egg Ca and CG release (& cell cycle resumption) will advance our knowledge of the role of Ca in fertilization.
Aim 2) The biochemical identification of the egg protein(s) modifying ZP3 is an important step in elucidating the molecular mechanism responsible for the mammalian block to polyspermy. This research may lead to a new non-invasive egg activation assay of released CG proteins (for optimizing or monitoring animal and human in vitro fertilization).
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