Yamamuro Tadashi, Kano Kiyoshi, Naito Kunihiko
Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Biol Reprod. 2008 Dec;79(6):1202-9. doi: 10.1095/biolreprod.108.070326. Epub 2008 Aug 27.
Cell division cycle 20 (CDC20) and fizzy/cell division cycle 20 related 1 (FZR1) are activators of the anaphase-promoting complex (APC), which ubiquitinates M-phase regulating proteins, such as cyclin B and securin, and induces their degradation. In the present study, porcine CDC20 and FZR1 were cloned by reverse transcriptase-polymerase chain reaction, and their functions in the meiotic maturation of porcine oocytes were analyzed. FZR1 was readily detected in porcine immature oocytes by immunoblotting, but its levels decreased substantially during maturation. In contrast, CDC20 levels rose during oocyte maturation and were highest by the second meiotic metaphase. The inhibition of CDC20 expression by the injection of CDC20 antisense RNA induced the meiotic arrest at the first meiotic metaphase (M1) and the accumulation of a large amount of cyclin B. On the other hand, the inhibition of FZR1 expression accelerated cyclin B accumulation and the start of germinal vesicle breakdown (GVBD), but did not affect the exit from M1. Conversely, the overexpression of FZR1 by the injection of FZR1 mRNA suppressed the cyclin B accumulation and retarded GVBD. Surprisingly, the injection of CDC20 mRNA into the immature oocytes could not increase CDC20 expression, but increased cyclin B accumulation and accelerated the meiotic progression. As CDC20 is a substrate of APC (FZR1), CDC20 might have competed with cyclin B and inhibited the FZR1 function. These results suggest that porcine FZR1 and CDC20 work on the maintenance of meiotic arrest at the first meiotic prophase and on the exit from M1, respectively, and that their functional phases are strictly distinguished during porcine oocyte maturation.
细胞分裂周期20（CDC20）和类Fizzy/细胞分裂周期20相关蛋白1（FZR1）是后期促进复合体（APC）的激活因子，该复合体可使M期调节蛋白（如细胞周期蛋白B和分离酶）发生泛素化，并诱导其降解。在本研究中，通过逆转录-聚合酶链反应克隆了猪CDC20和FZR1，并分析了它们在猪卵母细胞减数分裂成熟过程中的功能。通过免疫印迹法在猪未成熟卵母细胞中很容易检测到FZR1，但其水平在成熟过程中大幅下降。相反，CDC20水平在卵母细胞成熟过程中升高，并在第二次减数分裂中期达到最高。通过注射CDC20反义RNA抑制CDC20表达会诱导减数分裂停滞在第一次减数分裂中期（M1），并积累大量细胞周期蛋白B。另一方面，抑制FZR1表达会加速细胞周期蛋白B的积累和生发泡破裂（GVBD）的开始，但不影响从M1期退出。相反，通过注射FZR1 mRNA过表达FZR1会抑制细胞周期蛋白B的积累并延缓GVBD。令人惊讶的是，将CDC20 mRNA注射到未成熟卵母细胞中并不能增加CDC20表达，但会增加细胞周期蛋白B的积累并加速减数分裂进程。由于CDC20是APC（FZR1）的底物，CDC20可能与细胞周期蛋白B竞争并抑制FZR1的功能。这些结果表明，猪FZR1和CDC20分别在第一次减数分裂前期维持减数分裂停滞和从M1期退出过程中发挥作用，并且它们的功能阶段在猪卵母细胞成熟过程中严格区分。
