Introduction of Cyclin B Induces Activation of the Maturation-promoting Factor and Breakdown of Germinal Vesicle in Growing Zebrafish Oocytes Unresponsive to the Maturation-inducing Hormone

When treated with 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), a natural maturation-inducing hormone in fishes, fully grown zebrafish oocytes are induced to mature via the activation of the maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit). In contrast, 17alpha,20beta-DP is unable to induce growing (previtellogenic and vitellogenic) oocytes to mature. To know the reason growing oocytes fail to mature upon 17alpha,20beta-DP treatment, we investigated changes in the components of machinery responsible for MPF activation during zebrafish oogenesis. Immunoblotting experiments using monoclonal antibodies against cdc2, cyclin B, and cdk7 (an activator of cdc2) have revealed that the concentrations of cdc2 and cdk7 are almost constant during oogenesis. Cyclin B was present in mature oocytes but absent in growing and fully grown immature oocytes. These results, which are identical to those in goldfish, strongly suggest that cyclin B is synthesized from stored (masked) mRNA after 17alpha,20beta-DP stimulation and that its binding to the preexisting cdc2 allows cdk7 to activate MPF. Microinjection of cyclin B protein induced MPF activation and germinal vesicle breakdown in growing oocytes, as well as in fully grown oocytes, indicating that cdk7 present in growing oocytes is already active. Northern blot analysis revealed the presence of cyclin B mRNA in both previtellogenic and fully grown oocytes. These results indicate that, as in fully grown oocytes, growing oocytes are already equipped with the catalytic subunit of MPF (cdc2) and its activator (cdk7) and that the appearance of the regulatory subunit of MPF (cyclin B) is sufficient for initiating maturation. Therefore, the unresponsiveness of growing oocytes to 17alpha,20beta-DP is attributable to a deficiency in the processes leading to cyclin B synthesis, which include 17alpha,20beta-DP reception on the oocyte surface, subsequent signal transduction pathways, and unmasking the stored cyclin B mRNA.