| Like most other vertebrate oocytes, oocytes from Xenopus laevis are physiologically arrested at the first meiotic prophase. Re-initiation of meiosis, or oocyte maturation, is triggered by progesterone. Progesterone-induced oocyte maturation is thought to involve inhibition of cAMP-dependent protein kinase (protein kinase A or PKA). However, changes in PKA activity in live oocytes have never been demonstrated. I have developed a novel approach to monitor endogenous PKA activity in live oocytes, by expressing a PKA-specific substrate in oocytes followed by monitoring its PKA phosphorylation status during progesterone-induced oocyte maturation. I demonstrate that PKA is fully activated in prophase oocytes and that progesterone causes a rapid and permanent inhibition of PKA (Chapter 3). Utilizing this PKA activity indicator in live oocytes, I further demonstrate that progesterone-induced oocyte maturation requires persistent inhibition of PKA such that critical maturation-specific proteins can be synthesized and accumulated to threshold levels necessary for activation of maturation promoting factor (MPF) (chapter 4). In addition to analyzing PKA activity dynamics during oocyte maturation, I have also provided the first biochemical evidence supporting the notion that in prophase oocytes, a constitutively activated G protein coupled receptor system is responsible for elevated cAMP and the high levels of PKA activity (chapter 2). |
