Behavior of the components of maturation-promoting factor, cdc2 kinase and cyclin B, during oocyte maturation of goldfish.
We examined the changes that occurred in the two components of maturation-promoting factor (MPF), cdc2 kinase and cyclin B, during oocyte maturation in goldfish, using monoclonal antibodies against the C-terminal sequence of goldfish cdc2 kinase and Escherichia coli-produced full-length goldfish cyclin B. Immature oocytes contained a 35-kDa inactive cdc2 kinase. In addition to the 35-kDa form, a 34-kDa active cdc2 kinase was detected in oocytes undergoing germinal vesicle breakdown (GVBD). Cyclin B was absent in immature oocytes and appeared just before GVBD, coinciding exactly with the appearance of the 34-kDa active cdc2 kinase. Precipitation with p13suc1 beads and anticyclin B antibody revealed that cyclin B formed a complex with cdc2 kinase as soon as it appeared. MPF activation was induced by 1 ng cyclin B after introduction into immature oocytes or oocyte extracts. This corresponds to the amount of cyclin B found in mature oocytes (the concentration in the oocyte is 2 micrograms/ml). These results suggest that MPF activation in fish oocytes is induced by complex formation with preexisting cdc2 kinase and newly synthesized cyclin B during oocyte maturation, a situation differing from that in Xenopus and starfish, in which the cdc2 kinase-cyclin B complex is already present in immature oocytes. Unlike that in Xenopus, an inhibition of protein synthesis in unfertilized mature goldfish oocytes caused a decrease in the cdc2 kinase activity/cyclin B protein level and led to a progression from meiotic metaphase to meiotic anaphase. This result indicates that the mechanisms of maintaining MPF activity in mature goldfish oocytes differ from those in Xenopus.
Katsu
Y
Y
Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki, Japan.
Yamashita
M
M
Kajiura
H
H
Nagahama
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006054:Goldfish; D016200:Maturation-Promoting Factor / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000254:growth & development / Q000378:metabolism*
D016213:Cyclins; D003513:Cycloheximide; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor