The amphibian oocyte cell cycle is blocked at the first meiotic prophase until released by a developmental process known as maturation. Maturation is initiated by the action of the hormone progesterone and is associated with a period of intense protein phosphorylation. Unstimulated oocytes can be induced to undergo maturation, in vitro, by the microinjection of the regulatory subunit of cAMP-dependent protein kinase or an inhibitor of the kinase. These results suggest the involvement of protein phophorylation - dephosphorylation in the maturation process. We will examine the role of protein phosphorylation in oocyte maturation using the technique of photoaffintiy labeling. Photoaffinity analogues of the nucleotides ATP, GTP, cyclic AMP and cyclic GMP will be used to determine the protein phosphorylation and nucleotide binding profiles during maturation. These profiles will be established using gel electrophoresis and autoradiography. Proteins which appear to be involved with the maturation process will be assayed using microinjection techniques and purified using both traditional techniques and high performance liquid chromatography. Our ultimate goal is to define the machanism by which protein phosphorylation - dephosphorylation processes regulate oocyte maturation. Furthermore, since cultured mammalian cells synchronized in mitosis contain a factor which can induce oocyte maturation when microinjected, we envision these studies as a model for general cell cycle regulation.
