Cyclin B in fish oocytes: its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2 activation.
Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [35S]methionine, indicating its de novo synthesis. Introduction of E. coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17 alpha, 20 beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.
Hirai
T
T
Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Japan.
Yamashita
M
M
Yoshikuni
M
M
Lou
Y H
YH
Nagahama
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002474:Cell-Free System; D016213:Cyclins / Q000737:chemistry*; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli; D006054:Goldfish / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D009866:Oogenesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D016213:Cyclins; D011993:Recombinant Fusion Proteins; D004247:DNA; D016203:CDC2 Protein Kinase