Abstract
17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-DP) was identified as maturation-inducing hormone (MIH) in several teleost fishes. In goldfish ( Carassius auratus), 17α,20β-DP induces oocyte maturation by stimulating the de novo synthesis of cyclin B, a regulatory subunit of maturation-promoting factor (MPF). In this study, we examined the control mechanisms of 17α,20β-DP-induced de novo synthesis of cyclin B protein in oocytes, which is a prerequisite step for MPF activation during oocyte maturation in goldfish. Cycloheximide-treated oocytes failed to undergo meiotic maturation in response to 17α,20β-DP; in this group neither cyclin B nor 34-kDa active cdc2 was detectable in oocytes. In contrast, oocytes exposed to actinomycin D plus 17α,20β-DP or 17α,20β-DP underwent maturation; in these groups both cyclin B and 34-kDa cdc2 were present. Northern blotting showed that cyclin B mRNA is present in both immature and mature oocytes. Sequence analysis revealed that goldfish cyclin B mRNA contains four copies of cytoplasmic polyadenylation element (CPE)-like motifs in the 3′ noncoding region, suggesting that the initiation of cyclin B synthesis during oocyte maturation may be controlled by the elongation of poly (A) tail. We then examined the polyadenylation state of cyclin B mRNA during 17α,20β-DP-induced oocyte maturation by means of a PCR poly (A) test, and found that cyclin B mRNA is polyadenylated during oocyte maturation. Polyadenylation of cyclin B mRNA occurred at the same time of germinal vesicle breakdown. Furthermore, cordycepin, an inhibitor of poly (A) addition of mRNA, prevented 17α,20β-DP-induced oocyte maturation. These findings suggest that in goldfish oocytes, the synthesis of cyclin B protein is under translational control and that cytoplasmic 3′ poly(A) elongation is involved in 17α,20β-DP-induced translation of cyclin B mRNA.
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