LaRosa Cean, Downs Stephen M
Biology Department, Marquette University, 530 N 15th Street, Milwaukee, WI 53233, USA.
Mol Reprod Dev. 2005 Feb;70(2):235-45. doi: 10.1002/mrd.20200.
The present study was carried out to assess the possible role of mitogen-activated protein kinase (MAPK) in the meiosis-inducing action of the AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured 4 hr in Eagle's minimum essential medium containing dbcAMP plus increasing concentrations of AICAR or okadaic acid (OA). OA is a phosphatase inhibitor known to stimulate both meiotic maturation and MAPK activation and served as a positive control. Both OA and AICAR were potent inducers of meiotic resumption in mouse oocytes and brought about the phosphorylation (and thus, activation) of MAPK, but by different kinetics: MAPK phosphorylation preceded GVB in OA-treated oocytes, while that resulting from AICAR treatment appeared only after GVB. The MEK inhibitors, PD98059 and U0126, blocked the meiotic resumption induced by AICAR but not that induced by OA. Although the MEK inhibitors suppressed MAPK phosphorylation in both OA- and AICAR-treated oocytes, meiotic resumption was not causally linked to MAPK phosphorylation in either group. Furthermore, AICAR-induced meiotic resumption in Mos-null oocytes (which are unable to stimulate MAPK) was also abrogated by PD98059 treatment. A non-specific effect of the MEK inhibitors on AICAR accessibility to the oocyte was discounted by showing that they failed to suppress either nucleoside uptake or AICAR-stimulated phosphorylation of acetyl CoA carboxylase (ACC), a substrate of AMPK. The suppression of AICAR-induced maturation by MEK inhibitors must, therefore, be occurring by actions unrelated to MEK stimulation of MAPK; consequently, it would be prudent to consider this possible non-specific action of the inhibitors when they are used to block MAPK activation in mouse oocytes.
本研究旨在评估丝裂原活化蛋白激酶（MAPK）在AMP活化蛋白激酶（AMPK）激活剂5-氨基咪唑-4-甲酰胺-1-β-呋喃核糖苷（AICAR）诱导减数分裂中的可能作用。从经eCG预处理的未成熟小鼠获取卵丘细胞包裹的卵母细胞（CEO）或裸卵（DO），在含有dbcAMP以及浓度递增的AICAR或冈田酸（OA）的伊格尔氏最低必需培养基中培养4小时。OA是一种磷酸酶抑制剂，已知可刺激减数分裂成熟和MAPK激活，用作阳性对照。OA和AICAR都是小鼠卵母细胞减数分裂恢复的有效诱导剂，并导致MAPK的磷酸化（进而激活），但动力学不同：在OA处理的卵母细胞中，MAPK磷酸化先于GVB，而AICAR处理导致的MAPK磷酸化仅在GVB之后出现。MEK抑制剂PD98059和U0126阻断了AICAR诱导的减数分裂恢复，但未阻断OA诱导者。尽管MEK抑制剂抑制了OA和AICAR处理的卵母细胞中的MAPK磷酸化，但减数分裂恢复与两组中的MAPK磷酸化均无因果关系。此外，PD98059处理也消除了AICAR在Mos基因缺失的卵母细胞（无法刺激MAPK）中诱导的减数分裂恢复。MEK抑制剂对AICAR进入卵母细胞的非特异性作用被排除，因为它们未能抑制核苷摄取或AICAR刺激的乙酰辅酶A羧化酶（ACC，AMPK的底物）磷酸化。因此，MEK抑制剂对AICAR诱导的成熟的抑制作用必定是通过与MEK刺激MAPK无关的作用发生的；因此，在使用这些抑制剂阻断小鼠卵母细胞中的MAPK激活时，谨慎考虑其可能的非特异性作用是明智的。
