Fertilization marks a critical transition in the development of an organism. After lying dormant for months to years, fertilization awakens the oocyte, increasing its rate of metabolism and initiating its cell cycle leading to further embryonic development. How does sperm-egg contact trigger this awakening in the egg? Changes in intracellular ion concentrations are early steps in this activation mechanism. A transient [Ca2+]i increase and permanent pHi increase accompany egg activation in many species and treatments that activate eggs usually generate these ionic changes. Moreover, blocking the transient [Ca2+]i increase blocks egg 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 these ion concentration changes in the vertebrate egg? We will use the frog egg as a model system and test two main hypotheses. One states that the sperm introduces sufficient Ca2+ into the egg to trigger a process of Ca2+-induced Ca2+ release and the other is that the sperm triggers a wave of phosphatidyl inositol lipid hydrolysis that generates inositol trisphosphate (IP3) that in turn releases Ca2+ from the ER. We will also determine the mechanism for wave propagation in the frog egg. Specifically, we will determine if the wave spreads throughout the egg and if the mechanism of spread is the same in the cortical region near the plasma membrane as it is deep in the egg. Next, we will determine the mechanism of Ca2+ removal from the cytoplasm and how this might be linked to the permanent pHi increase that begins at the same time that [Ca2+]i is falling. After determining how the increase in [Ca2+]i is triggered and propagated. we will turn to pHi. There are several hypotheses we will test, including the possibility that the other byproduct of lipid hydrolysis, diacylglycerol, triggers the pHi increase by stimulating protein kinase C. Other ideas to be investigated include the possibility that the same mechanism pumping Ca2+ out of the cytoplasm also moves protons out. Finally, there is a good chance that the alkalinization is a byproduct of other metabolic changes occurring at activation. This research will clarify the relationships between the inositol lipid cascade, pHi and [Ca2+]i in a system that naturally undergoes physiological pHi and [Ca2+]i changes. Such information should prove valuable for the understanding of many cellular mechanisms which utilize pHi and/ir [Ca2+]i as regulatory agents.
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