Molecular Cloning and Immunological Analysis of Goldfish Cyclin A during Oocyte Maturation

Abstract

Cyclin A belongs to a family of proteins involved in the regulation of the eukaryotic cell cycle. Although cyclin A is thought to be involved in the regulation of both S and M phase, its exact role in the cell cycle, especially in the meiotic cycle (oocyte maturation), is uncertain. We isolated cyclin A cDNA clones from a goldfish oocyte cDNA library. Monoclonal antibody raised against bacterially produced goldfish cyclin A recognized a 47-kDa protein that disappeared after egg activation. Unlike goldfish cyclin B, which is absent in immature oocytes, cyclin A was already present in immature oocytes and its protein level did not change remarkably during oocyte maturation. These results differ from the finding in Xenopus, in which cyclin A is absent, but cyclin B is present, in immature oocytes. Goldfish cyclin A was associated with cdc2 kinase in mature oocytes, but not with cdk2. Recombinant cyclin A bound to and activated cdc2 in a cell-free system, but cyclin A and cdk2 binding was not observed. The kinase activity of cyclin A-cdc2 was undetectable in immature oocytes and first appeared at about the time of germinal vesicle breakdown (GVBD). In contrast to the cyclin B-cdc2 activity that corresponded to the occurrence of GVBD, cyclin A-cdc2 activity increased only slightly until GVBD was completed and increased drastically after the completion of the first meiotic division. Furthermore, microinjection of cyclin A mRNA into immature oocytes did not cause GVBD; however, microinjection of cyclin B mRNA did. These results suggest that cyclin A-cdc2 kinase and cyclin B-cdc2 kinase play different roles in controlling oocyte maturation. The roles of cyclin A in the rapid activation of cyclin B-cdc2 kinase at meiosis I and II transition and in the maintenance of high maturation-promoting factor activity in mature unfertilized eggs are discussed.