Kondo T, Yanagawa T, Yoshida N, Yamashita M
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Dev Biol. 1997 Oct 1;190(1):142-52. doi: 10.1006/dbio.1997.8673.
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.
用鱼类天然的成熟诱导激素17α,20β - 二羟基 - 4 - 孕烯 - 3 - 酮（17α,20β - DP）处理时，完全成熟的斑马鱼卵母细胞通过成熟促进因子（MPF）的激活被诱导成熟，MPF由cdc2（催化亚基）和细胞周期蛋白B（调节亚基）组成。相比之下，17α,20β - DP无法诱导正在生长的（卵黄生成前期和卵黄生成期）卵母细胞成熟。为了了解正在生长的卵母细胞在17α,20β - DP处理后不能成熟的原因，我们研究了斑马鱼卵子发生过程中负责MPF激活的机制成分的变化。使用针对cdc2、细胞周期蛋白B和cdk7（cdc2的激活剂）的单克隆抗体进行的免疫印迹实验表明，cdc2和cdk7的浓度在卵子发生过程中几乎保持恒定。细胞周期蛋白B存在于成熟卵母细胞中，但在正在生长的和完全成熟的未成熟卵母细胞中不存在。这些与金鱼中结果相同的结果强烈表明，细胞周期蛋白B是在17α,20β - DP刺激后从储存的（被掩盖的）mRNA合成的，并且它与预先存在的cdc2结合使cdk7能够激活MPF。向正在生长的卵母细胞以及完全成熟的卵母细胞中显微注射细胞周期蛋白B蛋白可诱导MPF激活和生发泡破裂，这表明正在生长的卵母细胞中存在的cdk7已经具有活性。Northern印迹分析揭示了在卵黄生成前期和完全成熟的卵母细胞中均存在细胞周期蛋白B mRNA。这些结果表明，与完全成熟的卵母细胞一样，正在生长的卵母细胞已经具备MPF的催化亚基（cdc2）及其激活剂（cdk7），并且MPF调节亚基（细胞周期蛋白B）的出现足以启动成熟。因此，正在生长的卵母细胞对17α,20β - DP无反应归因于导致细胞周期蛋白B合成的过程存在缺陷，这些过程包括卵母细胞表面的17α,20β - DP接收、随后的信号转导途径以及解开储存的细胞周期蛋白B mRNA的掩盖。
