【作者】 权赫梅；

【导师】 杨佩满；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 以小鼠和猪为实验动物,主要采用体外培养、药物处理、激光共聚焦显微术、免疫蛋白印迹以及抗体注射等方法研究了PKC在卵母细胞减数分裂、受精和胚胎早期发育过程中的表达,分布和作用。并研究卵母细胞成熟之前GV期精子的穿透是否影响MAPK磷酸化和减数分裂细胞周期进程。脊椎动物卵母细胞的减数分裂和受精过程受到多种蛋白激酶的调节。近年来对于卵母细胞成熟、活化和受精的分子机制研究取得了很大进步,蛋白激酶C(PKC)是广泛分布在真核生物体内的丝/苏氨酸蛋白激酶家族,有证据证明PKC在哺乳动物卵母细胞减数分裂成熟和活化过程中起着关键性的作用。然而很少了解PKC和PKC亚形在上述过程中所发挥的作用机制。我们揭示了小鼠卵和早期胚胎的:1)减数分裂和有丝分裂过程中PKC的调节机制;2)经典型PKC亚类在这些过程中的作用;3)从卵母细胞GV期发育到胚胎的胚泡期PKCα亚形的亚细胞定位。研究结果显示PKC激活剂PMA不仅依靠经典型PKC也依靠其他PKC亚形抑制小鼠裸卵减数分裂的恢复。GVBD以后发生的PKC活化作用导致MAPK磷酸化的抑制并使细胞周期阻滞在MI期。PKC活化也引起受精卵第二极体排出的失败。PKC(亚形集中在MI期前浓缩的染色体周围,也位于受精卵原核或早期胚胎分裂球的核。受精卵中原核的形成被PKC抑制剂钙感光蛋白C而不是被cPKC –特效抑制剂Go 6976抑制。PMA引起PKC活化时间延长导致早期胚胎的分裂被阻断。所有这些结果都说明PKC在小鼠卵母细胞和胚胎的细胞周期进程中具有多重功能。不同成熟阶段的猪卵与精子融合后体外培养发现:(1)不同成熟阶段的猪卵母细胞都可被精子穿透;(2)精子穿透不影响减数分裂细胞周期进程;(3)精子穿透GV期卵母细胞和正在成熟的卵母细胞不改变MAPK磷酸化:(4)已受精的前中期I(pre-MI)和中期I(MI)卵母细胞的MAPK有活性,MAPK不像MII卵母细<WP=6>胞那样受精后发生磷酸化。结果显示卵母细胞成熟之前精子的穿透并不影响MAPK磷酸化和减数分裂进程,卵母细胞成熟后才获得失活MAPK的机制。更多还原

【Abstract】 The meiosis and fertilization of vertebrate oocyte are extensively regulated by various protein kinases. Recently, a great progress has been achieved in the studies on the molecular mechanisms of oocyte maturation, activation and fertilization. The cooperation of these protein kinases is essential to the development and fertilization of the oocyte. Protein kinase C (PKC) is a family of ser/thr protein kinases widely distributed in eukaryotes. There is evidence that PKC plays key roles in meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we revealed in mouse eggs and early embryos: 1) the effects of PKC modulation on meiotic and mitotic cell cycle progression; 2) the functional importance of classical PKC subclasses in these processes; and 3) the subcellular localization of PKC( isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicated that PKC activator PMA inhibited meiotic resumption of denuded mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after GVBD leads to inhibition of MAPK phosphorylation and cell cycle arrest at the MI stage. PKC activation also caused failure of the second polar body emission after fertilization. PKC( isoform was typically concentrated around the condensed chromosomes before the MI stage, but localized in the pronuclei of zygotes and the nuclei of blastomeres of early embryos. Pronucleus formation in fertilized eggs was inhibited by PKC inhibitor calphostin C, but not by cPKC-specific inhibitor Go 6976. Cleavages of early embryos were blocked after prolonged PKC activation by PMA. All these results suggest that PKC plays multiple functional roles in cell cycle progression of mouse oocytes and embryos.Pig oocytes cultured in vitro for some time were inseminated by frozen–thawed <WP=8>ejaculated sperm. At specified times after insemination, sperm penetration, cell cycle progression and mitogen-activated protein kinase (MAPK) phosphorylation were evaluated. It was shown that: (1) oocytes at various maturational stages could be penetrated by sperm; (2) sperm penetration did not affect meiotic cell cycle progression; (3) sperm penetration of germinal vesicle (GV) oocytes and maturing oocytes did not alter MAPK phosphorylation; and (4) when premetaphase I (pre-MI) and metaphase I (MI) oocytes, in which MAPK was activated, were fertilised, no evident MAPK dephosphorylation was detected as in metaphase II oocytes. The data suggest that sperm penetration before oocyte maturation does not affect MAPK phosphorylation and that the machinery inactivating MAPK upon fertilisation is not developed in maturing (pre-MI to MI) oocytes.更多还原