Fertilization marks a critical transition in the development of an organism. After lying dormant for months to years, fertilization awakens the egg, increasing its rate of metabolism and initiates the cell cycle leading to further development. How does sperm-egg contact trigger this awakening in the egg? Changes in intracellular ion concentrations are critical early steps in this activation mechanism. A transient increase in intracellular calcium ([Ca2+]i) accompanies egg activation in all plant and animal species investigated, and treatments that activate eggs usually generate this ionic change. Moreover, blocking the transient [Ca2+]i increase blocks activation in all species studied, so this change is critical for the activation of development. The central question we are asking is how does the sperm trigger this increase in [Ca2+]i in the vertebrate egg? We will use the frog egg as a model system because of the large numbers of eggs and molecular tools available for this system. We will test the two most popular hypotheses for egg activation, the sperm receptor hypothesis and the diffusible factor hypothesis. We have recent evidence in support of the former. We have cloned a sperm surface protein, xMDC16, that appears to play a key role in fertilization in that an 8 amino acid peptide from the disintegrin domain of this protein can block fertilization at concentrations of 500 muM or higher. We have also found that this peptide can activate eggs when applied locally to the egg. This suggests the possible involvement of a sperm binding integrin on the egg's surface. Additional support for the involvement of an integrin is our observation of the stimulation of focal adhesion kinase (FAK) phosphorylation at fertilization along with the phosphorylation of three members of the Src-family of non-receptor tyrosine kinases. We propose to identify the signaling molecules involved in the cascade leading to Ca2+ release; purify the sperm receptor on the egg's plasma membrane; and to determine if the sperm initiates the Ca2+ release cascade via a diffusible factor or via a signal transducing receptor in the egg membrane.
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