Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburska 89, 277 21 Libechov, Czech Republic.
Reproduction. 2009 Oct;138(4):645-54. doi: 10.1530/REP-08-0461. Epub 2009 Jul 24.
The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) matured in vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 microM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.
本研究旨在探讨丝氨酸/苏氨酸蛋白激酶 AKT(也称为蛋白激酶 B)在体外成熟猪去卵丘卵母细胞(DO)减数分裂中的作用。Western blot 分析显示,两个主要的 AKT 磷酸化位点,Ser473 和 Thr308,在减数分裂的不同阶段发生磷酸化。在刚分离的生发泡(GV)期 DO 中,Ser473 已经发生磷酸化。在卵母细胞成熟开始后,Ser473 的磷酸化强度增加,但在 DO 发生 GV 破裂(GVBD)时急剧下降,并在 MⅠ期和 MⅡ期保持低水平。相比之下,Thr308 的磷酸化在 GVBD 时增加,并在 MⅠ期达到最大值。AKT 活性在 GVBD 时达到峰值,在 MⅠ期下降。为了评估 AKT 在减数分裂中的作用,将猪 DO 在 50μM SH-6 中培养,SH-6 是 AKT 的特异性抑制剂。在 SH-6 处理的 DO 中,GVBD 未被抑制;相反,减数分裂恢复的速度明显加快。Ser473 磷酸化的动力学没有受到影响;然而,Thr308 的磷酸化减少,AKT 活性在 GVBD 时降低,减数分裂停滞在早期 MⅠ期。此外,当 SH-6 处理的 DO 发生 GVBD 时,周期蛋白依赖性激酶 1(CDK1)和 MAP 激酶的活性下降,表明 AKT 活性参与了 CDK1 和 MAP 激酶的调节。这些结果表明,AKT 的活性对于诱导猪卵母细胞 GVBD 不是必需的,但在减数分裂向 MⅠ/ MⅡ期的进展中起着重要作用。