Shimaoka Takuma, Nishimura Takanori, Kano Kiyoshi, Naito Kunihiko
Laboratory of Applied Genetics, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Cell Cycle. 2009 Aug;8(15):2375-84. doi: 10.4161/cc.8.15.9073. Epub 2009 Aug 20.
Porcine immature oocytes require protein synthesis for meiotic resumption, thus the importance of Cdc2 inhibitory phosphorylation in their meiotic arrest remains controversial. We examined the involvement of Cdc2 phosphorylation in the meiotic arrest of porcine oocytes with a special focus on Wee1B, an oocyte-specific Wee1 family member recently reported in mouse oocytes. We cloned a Wee1B homologue of pig by RT-PCR followed by 5'- and 3'-RACE. Overexpression of pigWee1B in porcine immature oocytes by the injection of pigWee1B mRNA almost completely blocked the germinal vesicle breakdown (GVBD) under the low cAMP concentration, which could not block their spontaneous meiotic resumption by itself. The MPF activation and cyclin B synthesis were inhibited in these oocytes. Conversely, downregulation of pigWee1B expression by the injection of specific antisense mRNA induced GVBD in the oocytes, the spontaneous meiotic resumption of which was blocked by the high concentration of cAMP (dbcAMP). In these oocytes, the MPF activity was elevated and cyclin B was accumulated. Downregulation of pigMyt1, another Wee1 family member, could not induce the GVBD under the same condition. The inhibition of tyrosine phosphatase by vanadate blocked the GVBD even in the pigWee1B-downregulated oocytes. These results suggest that the inhibitory phosphorylation of CDC2, which is catalyzed by pigWee1B, but not pigMyt1, is involved in the meiotic arrest of porcine oocytes, and that the inactivation of Wee1B in combination with the phosphatase activation induces the conversion of pre-MPF to the active MPF and starts the cyclin B synthesis, follwed by a further increase of MPF and meiotic resumption.
猪未成熟卵母细胞减数分裂恢复需要蛋白质合成，因此Cdc2抑制性磷酸化在其减数分裂阻滞中的重要性仍存在争议。我们研究了Cdc2磷酸化在猪卵母细胞减数分裂阻滞中的作用，特别关注Wee1B，它是最近在小鼠卵母细胞中报道的卵母细胞特异性Wee1家族成员。我们通过RT-PCR，随后进行5'-和3'-RACE克隆了猪的Wee1B同源物。通过注射猪Wee1B mRNA在猪未成熟卵母细胞中过表达猪Wee1B，在低cAMP浓度下几乎完全阻断了生发泡破裂（GVBD），而低cAMP浓度本身并不能阻断其自发减数分裂恢复。这些卵母细胞中的MPF激活和细胞周期蛋白B合成受到抑制。相反，通过注射特异性反义mRNA下调猪Wee1B表达可诱导卵母细胞发生GVBD，其自发减数分裂恢复被高浓度cAMP（dbcAMP）阻断。在这些卵母细胞中，MPF活性升高，细胞周期蛋白B积累。另一个Wee1家族成员猪Myt1的下调在相同条件下不能诱导GVBD。钒酸盐对酪氨酸磷酸酶的抑制即使在猪Wee1B下调的卵母细胞中也能阻断GVBD。这些结果表明，由猪Wee1B而非猪Myt1催化的CDC2抑制性磷酸化参与了猪卵母细胞的减数分裂阻滞，并且Wee1B失活与磷酸酶激活相结合可诱导前MPF转化为活性MPF并启动细胞周期蛋白B合成，随后MPF进一步增加并导致减数分裂恢复。
