Src家族激酶在小鼠和大鼠卵母细胞减数分裂成熟过程中的定位及功能研究

【作者】 郑克刚；

【导师】 李云龙；

【作者基本信息】 山东师范大学 ， 动物学， 2007， 博士

【摘要】 哺乳动物卵母细胞在卵巢中停滞在第一次减数分裂前期的双线期,当卵母细胞受到促性腺激素的刺激后或者,从卵泡中释放出来后,会恢复减数分裂,发生生发泡破裂(germinal vesicle breakdown, GVBD)、继而染色质凝集、纺锤体组装、排出第一极体,发育到第二次减数分裂中期(Metaphase II, MII)再次阻滞。受精或孤雌活化后,卵子恢复第二次减数分裂并排出第二极体。减数分裂成熟的过程包含着合成细胞周期调控所需要的特殊的蛋白及通过磷酸化或去磷酸化来改变一些蛋白激酶的活性等分子事件。卵母细胞在减数分裂特定时期阻滞和恢复的机制研究一直是细胞生物学和发育生物学研究的热点之一。Src家族激酶(Src family kinases, SFKs)属于非受体酪氨酸蛋白激酶,由于它独特的空间构型及瞬间被激活的特性,正逐渐引起人们的关注。静息(或止)状态下,SFKs由于C末端酪氨酸残基的作用,使它们处于低活性状态或者说未活化状态,当C末端的酪氨酸残基去磷酸化,或者,由于Src同源结构域(Src homology,SH)2(SH2)和3(SH3),与其它的蛋白相互作用,SFKs的空间结构发生改变,使它的催化区域暴露出来,从而使该区域特殊位点上的酪氨酸发生自磷酸化,激活SFKs。人们对SFKs在体细胞有丝分裂过程中的活性变化和作用进行了大量的研究并且证实,SFKs参与了有丝分裂过程的调节。最近人们的研究发现,SFKs可能参与调整无脊椎动物(如棘皮动物、海鞘类动物)和脊椎动物(如非洲爪蟾、斑马鱼、大鼠)等动物卵子的活化过程和非洲爪蟾卵母细胞的成熟过程。然而,迄今为止,人们还没有对SFKs在哺乳动物卵母细胞减数分裂成熟过程中的作用进行研究。我们通过运用蛋白质免疫印迹、激光共聚焦显微术等技术研究了SFKs在小鼠、大鼠卵母细胞中的表达、亚细胞定位,并用药物处理的方法来研究SFKs在小鼠、大鼠卵母细胞成熟过程中的功能。在本实验中,我们选用了一种特殊的SFKs生理性抑制剂PP2,并选用细胞松弛素B(cytochalasin B, CB)(微丝聚合抑制剂)、秋水仙素(Colchicine)(微管组装阻断剂)来研究细胞骨架微丝、微管与SFKs之间的关系;同时,我们分别用A23187(Ca2+载体)和豆蔻酰佛波醇乙酯(phorbol 12-myristate 13-acetate,PMA,蛋白激酶C激活剂)来研究小鼠卵母细胞成熟过程中SFKs与Ca2+和蛋白激酶C(prorein kinase C,PKC)之间的关系。我们的研究发现:1、当小鼠卵母细胞从卵泡中释放出来半个小时后,SFKs开始出现在卵母细胞中,大约1小时后,SFKs被磷酸化激活;随着卵母细胞的成熟进程,无论是磷酸化的SFKs还是非磷酸化的SFKs在生发泡破裂前分别向核内和卵皮质区集中,的亚细胞定位都随之改变;当GVBD后,它们的亚细胞定位基本趋于稳定,均匀分布在卵胞质中。当SFKs的活性被抑制后,小鼠卵母细胞的GVBD率显著降低,发生了GVBD的卵母细胞的进一步发育被阻断,同时共聚焦显微镜检测结果表明,卵母细胞被阻滞在GVBD时期,并且不能成功的组装第一次减数分裂的纺锤体,不能发育到MII期和排出第一极体。在体外受精过程中,SFKs处于持续活化状态。当选用20μM PP2抑制SFKs活性后,微丝不能正常聚合;而用细胞松弛素B阻断微丝聚合后则发现SFKs的活性显著降低。这不仅说明微丝的聚合需要SFKs的参与,同时说明SFKs功能的发挥在一定程度上依赖于微丝的聚合。GV期的卵母细胞用蛋白激酶C的激活剂PMA及钙离子载体A23187处理30分钟后,检测不到磷酸化的SFKs在卵母细胞内的定位,说明Ca2+和PKC对SFKs的活性及定位没有影响。2、SFKs在大鼠卵母细胞的成熟过程中存在并都持续处于活化状态;抑制剂PP2处理结果表明,当用较高浓度的PP2抑制SFKs的活性后,大鼠卵母细胞的GVBD发生率受到显著影响。共聚焦显微镜结果显示,磷酸化的SFKs的亚细胞定位同微丝的亚细胞定位有一定的关联性。用100μM PP2处理GV期卵母细胞3小时后,微丝的定位及聚合没有发生改变;用5μM微丝解聚剂细胞松弛素B和10μg/ml的微管组装抑制剂秋水仙素(Colchicine)处理GV期卵母细胞10小时后,磷酸化的SFKs的亚细胞定位没有发生改变。这也同时表明SFKs在大鼠卵母细胞成熟过程中的作用不同于其在小鼠卵母细胞成熟过程中的作用。总之,通过我们的实验,首次研究了SFKs在哺乳动物(小鼠、大鼠)卵母细胞体外培养减数分裂成熟过程中的作用,可以得到如下结论:SFKs存在于小鼠和大鼠成熟过程中的卵母细胞内,对卵母细胞的减数分裂的成熟过程发挥着调控作用;SFKs可能参与了小鼠卵母细胞的活化过程的调节;SFKs在卵母细胞内功能的行使可能与细胞骨架微丝和微管相关联;蛋白激酶C和钙离子浓度的升高对于小鼠卵母细胞减数分裂成熟过程中SFKs的激活没有产生影响。更多还原

【Abstract】 Meiotic maturation of oocyte is a final step of oogenesis and a prerequisite process for the immature oocyte to become fertilization. In mammalian oocyte, meiosis resumes when the envelope of the germinal vesicle breakdown (GVBD), occurs a few hours after the luteinizing hormone (LH) surge in vivo, or oocyte releasing from the follicle in vitro. And then undergo a second arrest at metaphase II (MII), which is maintained until fertilization. Meiotic maturation involves synthesis of a series of specific proteins and changes in the phosphorylation state of kinase and phosphatase activity. Many of the components in the maturation induction pathway are still unknown. Kimura et al., (1996) indicated that protein tyrosine phosphorylation may be implicated in the regulation of mouse oocyte meiosis.The Src family kinase (SFKs) were first identified as transforming proteins encoded by oncogenic retroviruses. They are non-receptor tyrosine kinases and keep in a confirmation of low activity conformation by phosphorylation of C-terminal tyrosine residue. Upon dephosphorylation of C-terminal tyrosine residue or displacement of the Src homology regions 2 (SH2) and 3 (SH3) intramolecular interactions by other proteins, SFKs gains a relaxed conformation that allows autophosphorylation of tyrosine in the catalytic site and stabilizes the high activity state. Earlier Studies indicated that the different subcellular localizations of SFKs could be important for the regulation of specific cellular processes (reviewed by Bjorge et al., 2000). Previous studies showed the function of SFKs in the normal cell cycle control: As to the role of SFKs in oocytes, Spivack et al (1984) and Unger and Steele (1992) demonstrated the accelerated rate of progesterone-induced Xenopus oocyte maturation by microinjection of v-Src protein or by the expression of activated c-Src respectively. Furthermore, studies in echinoderm, ascidian, Xenopus, zebrafish and rat oocytes suggest that activation of an SFK(s) is required for oocyte activation. Numerous studies suggest that SFKs also function to regulate the actin cytoskeleton.We used western blot, confocal microscope immunofluorescence to study the in vitro dynamics of the subcellular distribution of SFK during the mouse oocyte meiotic maturation and evaluated the functions of SFK by PP2, a specific SFKs pharmacological inhibitor. Our results showed that nonphospho-SFK were absent in oocyte upon it was released from follicle. Nonphospho-SFK appeared in cytoplasm 0.5 h after the release of oocyte and were translocated to germinal vesicle before the onset of germinal vesicle breakdown (GVBD). After oocytes underwent GVBD, nonphospho-SFK accumulated in the same area as condensed chromosomes. In metaphase I and telophase I stage oocytes, nonphospho-SFK accumulated in the cortex and the cleavage furrow respectively in addition to distributing throughout the cytoplasm. In metaphase II oocytes, nonphospho-SFK concentrated around the aligned chromosomes. In contrast, phospho-SFK were absent in oocyte until 1 h after it was released from the follicle. Phospho-SFK accumulated in the germinal vesicle and the cortex in addition to their cytoplasm localization immediately prior to GVBD. After GVBD, phosphot-SFK distributed evenly in oocyte. In MII oocyte, phospho-SFK were localized throughout the cytoplasm and under the egg member. When the SFK activity was inhibited, oocyte failed to onset GVBD, MI entry and extrude the first polar body. Our research also shown The interaction between SFKs and F- actin .when the activity of SFKs were inhibited, F-actin were depolymerized, by contraries, The activity of SFKs were severely affected when inhibited F-actin polymerize. We also try to investigated the relationship between SFKs and protein kinase C (PKC) or Ca2+, we found that the activity of SFKs were not be affected when oocytes were treated by PMA or A23187. Furthermore, our study shown that SFKs keep its activity during in vitro fertilization (IVF).Then we used confocal microscope immunofluorescence, specific SFKs pharmacological inhibitor methods to evaluate whether SFKs have the same functions during meiotic maturation in rat oocytes. Our results shown that SFKs were localized under the cell membrane (the same area of cortex) and around the chromosome, and were keeping its activity during all the process of oocyte maturation. So the results in hinted that SFKs may be involved in modulation of meiotic maturation in rat oocytes. Our inhibitor methods data shown that 100μM PP2 had significant effect in oocytes GVBD, it was indicted that the activity of SFKs were necessary for GVBD of rat oocytes. The localization of SFKs was same as the localization of cytoskeleton in rat oocytes, so we suspected that the function of SFKs may be related with F-actin or microtubule. We choose 5μM cytochalasin B (an inhibitor of F-actin polymerize) and 10μg/ml Colchicine (an inhibitor of microtubule organization). Our data shown that the activity of SFKs were not affected when F-actin polymerize or microtubule organization were inhibited. Furthermore, it was not alike in mouse oocytes, when inhibited the activity of SFKs or the polymerization of F-actin during rat oocytes maturation, we did not find any change of the location of SFKs or F-actin. So SFKs may have different functions in rat oocytes than they have in mouse oocytes.In conclusion, our results indicated for the firs time that SFKs were localized in mouse and rat oocytes during meiotic maturation process, and the function of SFKs may be related with cytoskeleton such as F-actin or microtubule, and protein kinase C or Ca2+ had no effect on the activity of SFKs during the process in mouse oocytes.更多还原