@misc{oai:ir.soken.ac.jp:00001307,
 author = {勝, 義直 and カツ, ヨシナオ and KATSU, Yoshinao},
 month = {2016-02-17, 2016-02-17},
 note = {Oocyte maturation is a prerequisite for successful fertilization. This process in fish oocytes has been reported to be regulated by three major mediators: gonadotropin, maturation-inducing hormone (17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP, in fish) and maturation-promoting factor (MPF, a complex of cdc2 kinase and cyclin B). Cyclins have been reported to regulate the kinase activity of cdc2, leading to the activation and inactivation of MPF. The present study investigates the roles of cyclin A and B and the mechanisms of translational activation of maternal cyclin B mRNA during 17α, 20β-DP-induced oocyte maturation in goldfish (Carassius auratus). <br />  Western blot analysis was used to examine changes in the protein levels of cdc2 kinase and cyclin B during goldfish oocyte maturation induced in vitro with 17α, 20β-DP. Immature oocytes contained a 35-kDa cdc2. In addition to this protein, a 34-kDa cdc2 was detected in mature oocytes. The purified MPF contained the 34-kDa cdc2, but not 35-kDa cdc2. Thus, it is concluded that the 34- and 35-kDa cdc2 proteins are active and inactive forms, respectively. The 34-kDa active cdc2 appeared in accordance with the onset of germinal vesicle breakdown (GVBD). Cyclin B was absent in immature oocyte extracts and appeared when oocytes underwent GVBD, coinciding with the appearance of the 34-kDa active cdc2. Precipitation experiments with pl3suel and anti-cyclin B antibody revealed that cyclin B that appeared during oocyte maturation formed a complex with cdc2, as soon as it appeared. It is most likely that the 35-kDa inactive cdc2 preexisting in immature oocytes forms a complex with de novo synthesized cyclin B at first, then is immediately converted into the 34kDa active form, which triggers all changes that accompany oocyte maturation, such as GVBD, chromosome condensation and spindle formation. Introduction of E. coli-produced cyclin B into immature oocytes using microinjection induced oocyte maturation under condition of inhibited protein synthesis. These results strongly 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. <br />  Both in Xenopus and fish, unfertilized mature oocytes are arrested at the second meiotic metaphase until fertilized. In contrast to Xenopus, an inhibition of protein synthesis in unfertilized mature goldfish oocytes with cycloheximide caused a 30-50% decrease in the cdc2 kinase activity/cyclin B protein levels and an exit from meiotic metaphase-arrest. As compared with that occurring upon normal activation, the induced-decrease in the MPF activity was partial, and the cell cycle of the cycloheximide-treated oocytes was arrested again at the second meiotic anaphase. These results show that cdc2 kinase activity, cyclin B protein level, and cell cycle progression are closely linked. Furthermore, it is suggested that in addition to a difference in the mechanisms of MPF activation, the mechanisms of maintaining MPF activity in unfertilized mature goldfish oocytes differ from those in mature Xenopus oocytes. <br />  Although cyclin A is thought to be involved in the regulation of both S and M phase in eukaryotic cell cycle, its exact role in the cell cycle, especially in the meiotic cycle (oocyte maturation) is uncertain. To investigate the role of cyclin A in oocyte maturation, a goldfish cyclin A cDNA was cloned and antibodies against its product were produced. Unlike goldfish cyclin B that is absent in immature oocytes, cyclin A was already present in immature oocytes and its protein level was not remarkably changed during oocyte maturation. These observations differ from those of Xenopus oocytes, showing an undetectable amount of cyclin A and a large amount of stockpiled cyclin B at the onset of oocyte maturation. Thus, the behavior of goldfish cyclin A resembles that of Xenopus cyclin B, whereas that of goldfish cyclin B resembles Xenopus cyclin A. In the goldfish oocyte system, cyclin A binds to cdc2, but not cdk2, and that it activates cdc2 both in vivo and in vitro, raising the possibility that cyclin A plays a role in oocyte maturation. Changes in cyclin A-cdc2 and cyclin B-cdc2 kinase activity during oocyte maturation were also examined. Cyclin B-cdc2 kinase activity increases according to the occurrence of GVBD. Although the timing of the activation of the cyclin B-cdc2 and cyclin A-cdc2 complexes is almost the same, the rapid increase in cyclin A-cdc2 kinase activity occurs only after the completion of GVBD. It is possible that cyclin A-cdc2 kinase may play an important role in steps after GVBD; for example, the kinase may help the rapid activation of cyclin B-cdc2 kinase at meiosis I toll transition or play a part in the maintenance of high cyclin B-cdc2 kinase activity in mature unfertilized oocytes. It is concluded from these results that cyclin B-cdc2 kinase, but not cyclin A-cdc2 kinase, is important for oocyte maturation (especially GVBD). <br />  In the preceding sections, it was demonstrated that in goldfish oocytes de novo synthesis of cyclin B protein is required for the activation of MPF (cyclin B-cdc2 complex) during oocyte maturation. The next series of experiments were designed to investigate the mechanism of 17α,20β-DP-induced cyclin B synthesis. It was found that 17α,20β-DP-induced oocyte maturation was inhibited by a protein synthesis inhibitor (cycloheximide), but not by an RNA synthesis inhibitor (actinomycin D). Northern blot analysis showed that cyclin B mRNA is present in both immature and mature oocytes with no significant difference between them. Taken together, these results suggest that the synthesis of cyclin B protein is regulated at the translational level. Since it has long been recognized that the translational activity of maternal mRNAs generally correlates with changes in polyadenylation, the involvement of polyadenylation in cyclin B mRNA translation was examined. Examination of the 3'UTR of goldfish cyclin B mRNA revealed that it posesses a conseved sequence AAUAAA with four copies of cytoplasmic polyadenylation element (CPE, consensus U4-6A1-2U) motifs which are the cis-acting sequence that specifies cytoplasmic polyadenylation. A PCR poly (A) test revealed that poly (A) elongation occurs in goldfish cyclin B mRNA during oocyte maturation. However, it is noteworthy that this poly (A) addition occurred in oocytes which underwent GVBD, that is, after the appearance of cyclin B protein in oocytes, probably after MPF activation. Thus, it is concluded that cyclin B mRNA polyadenylation is not the major mechanism which is responsible for the initiation of cyclin B mRNA translation in goldfish oocytes. <br />  It has also been suggested that some RNA-binding proteins mediate translational process of stored maternal mRNAs. When poly (A)+RNA from goldfish immature oocytes was mixed with the reticulocyte lysate system, cyclin B could be synthesized, suggesting the translational ability of goldfish maternal cyclin B mRNA. Furthermore, the efficiency of cyclin B translation was much lower with extracts from immature oocytes than with those from mature oocytes. Thus, it is most likely that the initiation of cyclin B synthesis in goldfish oocytes is regulated by a translational inhibitory factor (RNA-binding protein). In this study, a cDNA clone encoding a goldfish Y box protein, which is known to sequester mRNA from translation in Xenopus oocytes, was isolated and partially characterized. The identification of the inhibitory factor and clarification of its role should elucidate the translational regulation of cyclin B synthesis during hormone-induced oocyte maturation in goldfish. Thus, the goldfish oocyte will provide a valuable model to gain better understanding of a basic mechanism for translational regulation of gene expression which has currently been accumulating as a primary regulatory mechanism in eukaryotic systems., application/pdf, 総研大甲第144号},
 title = {キンギョ(Carassius auratus)のサイクリンＡとＢ - 卵成熟過程における役割と合成機構 -},
 year = {}
}