Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection
Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection
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DOI：
10.1095/biolreprod.103.021519
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发表时间：
2004-01-01
影响因子：
3.6
通讯作者：
Tojo, H
中科院分区：
文献类型：
--
作者：
Kuroda, T;Naito, K;Tojo, H
本研究通过向猪卵母细胞内注射细胞周期蛋白B1（CB 1）和细胞周期蛋白B2（CB 2）的反义RNA（asRNA），研究了它们在猪卵母细胞成熟过程中的作用。首先，通过免疫印迹检测两种细胞周期蛋白B的蛋白水平，揭示未成熟卵母细胞仅具有CB 2，其水平相当于在第一中期卵母细胞中检测到的水平的1/20至1/40。两种细胞周期蛋白B的合成均始于胚泡破裂（GVBD）前后; CB 1和CB 2分别在第二中期和第一中期达到高峰。利用猪未成熟卵母细胞总RNA和人CB 2基因的引物，通过RT-PCR扩增，获得了一个与人CB 2基因同源性为88%的猪CB 2 cDNA片段。在体外制备CB 1和CB 2的特异性asRNA。然后将其中一种、另一种或两者注射到未成熟卵母细胞的细胞质中。CB 1 asRNA特异性地抑制CB 1的合成，注射的卵母细胞正常进行第一次减数分裂，但不能停止在第二次减数分裂中期。CB 2 asRNA特异性地抑制CB 2的合成，但对注射卵母细胞的成熟几乎没有影响。当同时注射CR 1和CB 2 asRNA时，细胞周期蛋白B的合成受到抑制，GVBD被显著抑制，但发生缓慢。这些结果表明，CB 1是哺乳动物卵母细胞成熟的主要调控分子，而CB 2只有辅助作用。他们还表明，在猪卵母细胞中，细胞周期蛋白B的合成本身不是GVBD诱导所必需的，但至少一种细胞周期蛋白B、CB 1或CB 2的合成在正常时间过程中是GVBD诱导所必需的。
The function of cyclin B1 (CB1) and cyclin B2 (CB2) during porcine oocyte maturation was investigated by injecting oocytes with their antisense RNAs (asRNAs). At first, protein levels of both cyclin Bs were examined by immunoblotting, revealing that immature oocytes had only CB2, at a level comparable to 1/20 to 1/40 of that detected in first metaphase oocytes. Both cyclin B syntheses were started around germinal vesicle breakdown (GVBD); CB1 and CB2 peaked at the second metaphase and first metaphase, respectively. We obtained a porcine CB2 cDNA fragment, which was 88% homologous with human CB2, by reverse-transcriptase polymerase chain reaction (RT-PCR) using total RNAs of immature porcine oocytes and a primer set of human CB2. Specific asRNAs of CB1 and CB2 were prepared in vitro. Then one, the other, or both were injected into the cytoplasm of immature oocytes. CB1 asRNA inhibited CB1 synthesis specifically; the injected oocytes underwent first meiosis normally but could not arrest at the second meiotic metaphase. CB2 asRNA inhibited CB2 synthesis specifically, but had almost no effect on the maturation of injected oocytes. When both CR1 and CB2 asRNAs were injected, synthesis of both cyclin Bs was inhibited, and GVBD was significantly suppressed but occurred slowly. These results suggest that CB1 is the principal molecule for regulation in mammalian oocyte maturation, whereas CB2 has only an accessory role. They also show that in porcine oocytes, cyclin B synthesis is not necessary for GVBD induction itself, but synthesis of at least one cyclin B, CB1 or CB2, is necessary for GVBD induction in a normal time course.