【作者】 范衡宇； 佟超； 陈大元； 孙青原；

【Author】 Heng-Yu Fan, Chao Tong, Da-Yuan Chen, Qing-Yuan Sun(State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy ofSciences, Beijing 100080, China)

【机构】 中国科学院动物研究所计划生育生殖生物学国家重点实验室；

【摘要】 丝裂原活化的蛋白激酶(mitogen-activated protein kinase,MAPK)在猪卵母细胞减数分裂成熟过程中被激活,但是它的生理底物还不很清楚。在非洲爪蟾和小鼠卵母细胞中,90kDa的核糖体S6蛋白激酶p90rsk是目前研究得最清楚的MAPK底物。本实验研究了p90rsk在猪卵母细胞减数分裂成熟、受精和孤雌激活过程中的表达、活化、亚细胞定位,及其可能作用。该激酶在生发泡(germinal vesicle,GV)期卵母细胞中通过不依赖MAPK的机制被部分磷酸化,但是它的充分磷酸化依赖于MAPK活性。在受精或电激活以后,p90rsk在原核形成稍前去磷酸化,与MAPK的失活同时发生。蛋白磷酸酶抑制剂冈田酸(OA)在减数分裂成熟过程中加速p90rsk磷酸化,并可以在已活化的卵子中诱导p90rsk的再磷酸化。MAPK上游激酶MEK的抑制剂U0126可以抑制卵丘包裹的卵母细胞和裸卵中MAPK和p90rsk的激活,但是只在卵丘包裹的卵母细胞中抑制生发泡破裂。活化状态的MAPK和p90rsk都存在于卵丘细胞中,U0126可以使二者发生去磷酸化。在处于MII期阻滞的MII期卵子中,U0126导致MAPK和p90rsk的失活,并使卵子的细胞周期向间期转化。激光共聚焦显微术研究表明,p90rsk均匀分布在GV期卵母细胞中,但在生发泡破裂以前向细胞核内集中。该分子在GVBD以后定位在减数分裂纺锤体上,并且在极体排放过程中集中在纺锤体的中板位置,与相应时期MAPK的亚细胞定位相一致。这些结果表明,p90rsk是MAPK的下游分子,并且在猪卵母细胞的细胞周期和纺锤体组装过程中发挥调节作用。在猪卵母细胞中,虽然MAPK和p90rsk活性不是裸卵发生自发减数分裂恢复所必需的,但卵丘细胞中该信号通路的激活是激素诱导的减数分裂恢复所必需的。更多还原

【Abstract】 Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90 kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. Protein phosphatase inhibitor okadaic acid (OA) accelerated the phosphorylation of p90rsk during meiotic maturation, and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK, MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced then: dephosphorylation. In Mil arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk distributed evenly in GV oocytes, but accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid-zone during emission of the polar bodies. All these results suggest that p90rsk is downstream to MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although activity of MAPK and p90rsk is not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.更多还原