小鼠和猪卵母细胞纺锤体组装和染色体分离与卵母细胞非整倍体形成的机制研究
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摘要
研究目的和意义
不孕不育和出生缺陷的根本原因是生殖细胞在发生和成熟过程中的异常造成受精和胚胎发育的失败或缺陷。近几十年来,人类妊娠中异常高发的非整倍体率二直是个顽固的遗传难题。据统计，人类大约有30%的卵子和9%的精子携带有不平衡的染色体；而且大龄女性排出的卵子发生染色体异常的风险大于50%；有7-10%的临床妊娠伴随染色体异常；自然流产胎儿中有60%为非整倍体所致,人工流产胎儿中5-10%为非整倍体。卵子高发的非整倍体严重影响了女性的生殖力。
卵子是生殖技术的基础,卵子的质量大大影响了受精、胚胎的早期发育和着床,因此卵子质量是不孕患者在辅助生殖中卵子受精,胚胎着床的保证。卵子也为合子和受精后前三天的胚胎提供RNA和蛋白,以营养和维持基因的稳定性,直至胚胎基因被激活’。有报道称辅助生殖中60%的人胚胎为非整倍体,大部分都不能着床,但是非整倍体胚胎一旦种植,就会发生早期发育停滞和流产。对大龄女性,反复着床失败、复发性流产和有非整倍体病史者行种植前的遗传学检查也证实非整倍体是出生缺陷,辅助生殖技术成功率低和复发性流产的主要原因之一。
母亲年龄是导致出生缺陷中非整倍体增加的主要病因。女性的生殖细胞一出生就意味着逐渐走向衰老,20±岁的年轻妇女排出的卵母细胞仅有2-3%发生染色体异常,而40岁时,这种风险增至30-35%。年龄增加了卵母细胞第一次和第二次减数分裂发生错误率。近30年来,女性第一次生育的年龄已经增加了3.6岁,导致了35岁后生育的女性明显增加,而大于35岁后生育的女性由于卵子染色体异常率高导致生育力明显下降。随着人类辅助生殖技术的迅速普及和成熟,由卵子非整倍体异常导致的着床失败所占比例逐渐增加,大龄妇女的生育是目前面临的一个挑战。而且有文献报道在辅助生殖体外受精后年轻女性发生复发性流产,反复非整倍体发生或种植失败也明显增加,这可能归因于超排卵中大剂量外源性的FSH应用和日益恶劣的环境污染,而且会影响胎儿期卵泡池中卵子的重组、联会和生长,增加成年后卵子对非整倍体的易感性,可能影响子代的配子质量2。
因此,研究女性卵子非整倍体的发病机制,减少非整倍体的发生,提高卵子质量是目前生殖领域最重要的课题之一。卵母细胞染色体不分离所致的非整倍体,直接导致胚胎停育或流产。正常的纺锤体组装是染色体正确分离的保障。其中纺锤体极的组装,纺锤体形状和长度也明显影响染色体的分离。只有通过蛋白激酶、磷酸化酶、动力蛋白、微管相关蛋白和纺锤体检验点蛋白等相互作用才能保证准确的纺锤体组装和染色体分离。目前对体细胞非整倍体发生机制的研究已非常深入,但是卵母细胞染色体分离异常所致的非整倍体发病机制不清。丝裂源活化蛋白激酶超家族(MAPK)信号通路是细胞内最重要的信号通路之一,它在许多重要的生物活动中起到至关重要的作用,如调控细胞增殖、分化以及细胞周期等。丝裂源活化蛋白激酶超家族是由经典的细胞外调节激酶(ERK), c-Jun或应激激活蛋白激酶(JNK)和p38组成,这些家族中的所有成员在真核细胞系中高度保守。ERK参加纺锤体组装和染色体的分离；另外作为MAPK的上游调控激酶MEK,它可能作为中心体蛋白,在微管组装,纺锤体极的聚合以及胞质不均等分裂中起作用。P38MAPK是MAPK家族中三个主要成员之一,它在调节细胞的生长分化；细胞的凋亡,增生以及细胞免疫反应和细胞骨架的重排中起到重要的作用。在体细胞中,p38a与MK2形成一个稳定的异源二聚体,在体内外磷酸化MK2并调节其功能。P38MAPK也参与前间期、G2/M细胞周期检验点功能,但p38MAPK是否是个有丝分裂的纺锤体组装检验点蛋白一直存在争议。而且本研究组前期已对MK2在小鼠卵母细胞减数分裂中的作用进行了探讨,发现其在纺锤体组装和同源染色体的分离中起到重要作用。在本研究中,我们首次对p38MAPK在小鼠卵母细胞减数分裂中的作用以及MK2在猪卵母细胞中的作用进行研究,以探讨其在哺乳动物卵母细胞非整倍体发生机制中的作用,为提高卵子质量、减少卵子和胚胎的非整倍性发生、辅助生殖技术的完善、生殖疾病防治和出生缺陷的减少奠定理论基础。
第一部分：P38αMAPK在小鼠卵母细胞减数分裂纺锤体组装和染色体分离中的作用
正常的纺锤体组装是确保染色体分离的重要前提。在纺锤体组装中,一定的细胞,其纺锤体的大小、形状和长度都相对固定,并受到动力蛋白、蛋白激酶和微管相关蛋白和极蛋白的调控。P38MAPK是MAPK家族中三个主要成员之一,在体细胞中已有研究认为其在细胞骨架的重排中起到重要作用。因减数分裂和有丝分裂的发生机制存在差异,减数分裂更为复杂,故各种蛋白在体细胞和卵母细胞中的作用不相同,本研究通过对P38MAPK在小鼠卵母细胞减数分裂中的作用探讨,丰富了我们对减数分裂这一重要细胞事件的了解,并为哺乳动物卵母细胞非整倍体的形成机制提供理论依据。
研究方法
取6周龄的ICR雌性小鼠的GV期卵母细胞在体外成熟培养,首先我们用免疫荧光对p38aMAPK进行准确定位；用免疫印迹检测p38aMAPK在小鼠各期卵母细胞中的蛋白表达水平；接着用免疫荧光检测Taxol处理后的卵母细胞p38aMAPK与微管组织中心及其相关蛋白的空间定位关系；进一步我们用p38aMAPK抑制剂、p38aMAPK mophornilo寡居核苷酸(p38a-MO)注射卵母细胞,抑制p38aMAPK的磷酸化或蛋白表达后研究其表型；同时我们用免疫共沉淀的方法检测了p38αMAPK和MK2的相互作用；接着我们用染色体铺片的方法证实MⅡ卵非整倍体的发生；最后用免疫荧光检测p38αMAPK功能破坏后对前中期卵母细胞中检验点蛋白BubRl的影响来探讨p38αMAPK对检验点蛋白的调控作用。
结果
1.磷酸化的p38αMAPK (p-p38α)在小鼠卵母细胞减数分裂成熟中的蛋白表达及亚细胞定位免疫印迹结果显示从生发泡期(GV)至中期(MI),p-p38a的蛋白表达量一直保持稳定,自后期到MⅡ期,蛋白表达量逐渐减少。p-p38a亚细胞定位：生化泡破裂期(GVBD)定位在染色体附近；前中期(Pre-MI),开始向纺锤体两极移动；MI期,p-p38a定位在纺锤体两极；中后期,没有检测到明显的p-p38a的信号；MⅡ期,p-p38a再次回到纺锤体极上。
2.p-p38a在小鼠卵母细胞减数分裂成熟中与中心体蛋白γ-tubulin定位关系在正常卵母细胞中生发泡破裂后,p-p38a在整个减数分裂成熟过程中都与γ-tubulin共定位；使用taxol微管聚合剂处理卵母细胞后,在前中期,中期和MⅡ期的异常纺锤体极,微管组装中心(MTOC)和胞浆星体的中心都能检测到p-p38a的信号,而且与γ-tubulin严格共定位。
3.评估破坏p-p38a功能后小鼠卵母细胞减数分裂中纺锤体组装和染色体排列p38a特异性的抑制剂SD203580处理GV卵后,影响了小鼠卵母细胞减数分裂中纺锤体组装和染色体排列。抑制剂组纺锤体异常率为70.5%(103/146),对照组18.54%(28/151),抑制剂组显著高对照组(χ2=81.430,P<0.001)；抑制剂组染色体错误排列率为61.64%(90/146),显著高于对照组13.25%(20/151),(χ2=74.563,P<0.001)。p38α-MO注射后影响了小鼠卵母细胞减数分裂中纺锤体组装和染色体排列,p38α-MO注射组中染色体异常率75.0%(129/172),与对照MO注射组(10.10%(19/188))比,染色体错排率显著升高(χ2=156.241,P<0.001)；此外,p38α-MO注射组中纺锤体异常率为74.4%(128/172),对照MO注射组21.3%(40/188),χ2=101.919,P<0.001,p38α-MO注射组显著高于对照组。
4.p38α与MK2在小鼠卵母细胞中的相互关系免疫共沉淀证实内源性的p38α与MK2能相互作用；p38α-MO注射后的卵母细胞中p-MK2蛋白的表达水平下降说明p38α能磷酸化MK2；p38α基因干扰后影响了p-MK2的定位,p-MK2从纺锤极和染色体上脱落,散落在卵的胞浆中,这一结果证实p38α能影响MK2的定位。
5.p38α对MI卵母细胞纺锤体极组装的影响MI卵母细胞纺锤体组装中典型的异常包括：极紊乱是纺锤体异常最突出的表现之一(实验组：57.89%&对照组：7.94%)；其它主要表现为一些单极(实验组：14.91%&对照组：3.97%)或另外的小极围绕着纺锤体或在极的附近,对照组与p38a-MO注射组比,Z=98.095,df=3,P<0.001(双侧),并可见γ-tubulin和标有γ-tubulin的MTOC星体分散在异常的纺锤体和MI卵的胞浆中。对p38a干扰后的卵母细胞行冷处理破坏非动粒微管后.,动粒微管完全不能聚合在纺锤体极韵两端。正常及Taxol处理后的卵母细胞,p-p38a与Plk1在MTOC和纺锤体极上严格共定位,p38a干扰后,Plk1完全从纺锤体极和染色体上脱落。
6.p38a或Eg5干扰及Eg5过表达后对纺锤体张力和长度的影响p38a干扰后,通过检测同源染色体动粒间的距离来评估其张力：p38a-MO注射组动粒间的距离为2.88±0.05-n,显著长于对照组(0.99±0.041μm),F=21.567,P<0.001,t=-47.985。纺锤体长度：p38α-MO注射组(39.5+3.37μm)显著长于对照组(23.3±3.02μm)(P<0.001)；Eg5过表达组纺锤体明显拉长(37.80±2.09μm),显著长于对照组(P<0.001)；Eg5过表达后与p38a-MO注射组纺锤体长度比,无统计学差异(P=0.163,P>0.05)；而Eg5功能缺失的卵却表现为单极纺锤体；同一个卵中同时注射p38a-MO和Eg5-MO后,双极纺锤体的形态和长度部分恢复(25.3±2.72μm),与对照组比无统计学差异(P=0.093,P>0.05),但形态不稳定。
7.p38α干扰后对Eg5和Dynein在纺锤体上分布的影响破坏Eg5和Dynein功能后,p38α在纺锤体上的定位无改变；正常卵母细胞：Eg5定位在微管和纺锤体极上；过表达Eg5后,Eg5均匀的定位在纺锤体的微管上,双极无表达；p38α基因干扰后：Eg5在极上的表达减少,而在纺锤体微管上的表达增加,尤其在纺锤体中板上的表达显著增加。
8.p38α功能破坏后,对MⅡ卵染色体排列和非整倍体率的评估以及对检验点蛋白BubR1的影响p38a缺失后并没有影响第一极体的排出(p38MO：78.2%,＆对照组：79.8%,n=196；χ2=0.123,P=0.730,P>0.05)；MⅡ卵表现明显的染色体排列异常,p38a-MO注射组MⅡ卵染色体异常率为65.70%(71/108),对照组MⅡ卵染色体异常率为7.9%(10/126),p38a-MO注射组染色体排列异常率显著高于对照组,χ2=85.850,P<0.001；MⅡ卵非整倍体率统计结果：p38a-MO注射组非整倍体率为42.4%(14/33),而对照组为4.8%(1/21)；p38a-MO组非整倍体率显著高于对照组,χ2=9.07,P=0.003,P<0.05。在注射p38α-MO后前中期卵母细胞中,大部分定位在卵母细胞上的BubR1从动粒上消失。
小结
1.p38α是小鼠卵母细胞无中心粒微管组装中心的一部分,能调节纺锤体的组装和染色体的排列p-p38α定位于纺锤体两极并与在微管组装中心和胞浆星体上的γ-tubulin和Plkl共定位,说明其是MTOC的一部分,参加了微管的组装和成核；p38α缺失导致了γ-tubulin和Plkl从纺锤体极上脱落,支持p38α参与微管成核,并能募集γ-tubulin到MTOC和参与纺锤体组装；p38α功能破坏导致了纺锤体形状异常和染色体排列紊乱,进一步支持p38α是减数分裂纺锤体组装中必不可少的蛋白激酶。p38α与MK2在小鼠卵母细胞中形成复合物,MK2是p38α的下游,p38α磷酸化其底物MK2,p38α通过小鼠卵母细胞减数分裂成熟中p38α-MK2信号通路参与纺锤体的组装和染色体的分离。
2.p38α能稳定纺锤体和纺锤体极。p38α功能缺失导致纺锤体组装异常,表现为多极,单极和极松散不粘合,有γ-tubulin信号的完整环形极从纺锤体上脱落,这一现象表明p38α能将极微管和无中心体的微管组装中心与微管的负极偶联；冷处理p38α-MO注射后MI卵破坏非动粒微管后,能更清晰的观察到动粒微管的负极末端不能粘合；这些结果都表明p38α能稳定纺锤体和纺锤体极。
3.p38α和Eg5在调节纺锤体的张力和长度时,相互拮抗,是一对反作用力量p38α功能的缺失导致了纺锤体的拉长和微管张力的增加,Eg5缺失导致了单极纺锤体,而过表达Eg5导致了纺锤体的拉长,和p38α功能缺失后的结果一致；P38α功能的缺失破坏了Eg5这一重要驱动蛋白在微管上的分布,故P38α和Eg5相互拮抗,通过影响驱动蛋白Eg5在纺锤体上的定位发挥调控纺锤体长度和大小的作用。
4.P38α调节小鼠卵母细胞纺锤体检验点。P38α功能缺失阻碍了BubR1在染色体动粒上的定位而损害了纺锤体检验点的功能,这是小鼠卵母细胞减数分裂中引起染色体排列异常且非整倍体率增加的重要原因。
第二部分：MK2在猪卵母细胞减数分裂纺锤体组装和染色体分离中的作用
正常的纺锤体组装是确保染色体分离的重要前提。在体细胞和小鼠卵母细胞中P38α和MK2形成一个复合体,P38α磷酸化MK2并影响MK2的定位,在小鼠卵母细胞中P38α通过P38α-MK2信号通路作用于其底物MK2调控纺锤体的组装和染色体的分离。我们研究组前期发现小鼠中MK2定位在纺锤体和染色体臂间,也在纺锤体组装和同源染色体的分离中起到重要作用。可是P38α在猪卵母细胞中不同于小鼠卵母细胞中的定位,其定位在猪卵母细胞的胞浆中,同是哺乳动物,却发现其有表达差异,这一结果提示我们进一步探讨MK2在猪卵母细胞减数分裂中的作用,并比较其和小鼠卵母细胞中的差异,以进一步完善MK2在哺乳动物卵母细胞减数分裂中的作用机制。
研究方法
取猪卵丘-卵母细胞复合物(COCs)在体外培养成熟,首先我们用MK2的抑制剂CMPD1处理COCs观察卵丘扩展以及对减数分裂进程的影响；接着用CMPD1处理裸卵,以观察MK2抑制剂CMPD1对裸卵减数分裂成熟的影响。用免疫荧光对MK2在猪卵母细胞的纺锤体和染色体上进行准确定位；其次用Taxol处理卵母细胞后用免疫荧光检测其与微管组装中心和Plk1、Crest和γ-tubulin的关系：再次我们用MK2抑制剂和p-MK2抗体分别注射卵母细胞,破坏MK2的磷酸化及其功能后研究其表型；最后用免疫荧光检测MK2功能破坏对Plkl定位的影响。
结果
1.MK2抑制剂CMPD1对猪卵丘-卵母细胞复合物卵丘扩展的影响猪卵丘细胞复合物在含有FSH的培养基中培养24h后,对照组卵丘扩展率为85.7%(281/328),然而高浓度的CMPD1(30μmol/L)几乎完全抑制了由FSH诱导的卵丘扩展(3.3%(8/242))；20μmol/L和10μmol/L组卵丘扩展率分别为：12.5%(30/240)＆24.39%(80/328)；χ2=546.22,P<0.001；故30,20,10和0μmol/L各处理组间有显著的差异,CMPD1呈剂量依赖性的抑制FSH诱导的卵丘扩展。
2.评价MK2抑制剂CMPD1对猪卵丘-卵母细胞复合物减数分裂成熟的影响MK2抑制剂CMPD1不影响猪卵母细胞自发的减数分裂恢复,但却阻碍了卵母细胞极体的排放。猪卵丘-卵母细胞复合物在含有FSH的培养基中培养42h,63.16%(60/95)的卵母细胞排放了第一极体,然而高浓度的CMPD1(30μmol/L)完全抑制了猪卵母细胞第一极体的排放；20pmol/L和10μmol/L的CMPD1的极体排放率为(6.25%(11/176)＆37.9%(50/132),χ2=179.045；P<0.001,故CMPD1呈剂量依赖性的抑制卵母细胞第一极体的排出。
3. MK2抑制剂CMPD1对猪裸卵减数分裂成熟的影响不同浓度CMPD1处理均未影响猪裸卵自发性减数分裂恢复；对照组裸卵极体排放率为54.46%(55/101),而高浓度的CMPD1(30μmol/L)处理完全抑制了猪卵母细胞第一极体的排放,极体排放率为0%(0/136);20μmol/L和10μmol/L CMPD1处理后第一极体排放率分别为5.41%(10/185)&19.48%(30/154)；χ2=150.055,P<0.001。10μmol/L CMPD1对猪裸卵(19.5%(30/154))第一极体排放的影响显著大于对COCs(37.9%(50/132)),χ2=11.94,P<0.001。
4.检测猪卵母细胞中MK2的亚细胞定位前中期和中期p-MK2沿着染色体臂定位于臂轴间隙和着丝粒区域,且与微管部分共定位,但是表达的面积小于α-tubulin;MⅡ期,p-MK2呈点状定位在动粒与姐妹染色体间,也就是姐妹染色体的着丝粒区。p-MK2与Plk1在微管上部分共定位,Plk1定位在染色体的动粒上,而p-MK2定位在染色体与Plk1之间。p-MK2与γ-tubulin在纺锤体上部分共定位。
5.观察MK2抑制剂CMPD1对猪卵母细胞纺锤体组装和染色体排列的影响CMPD1处理组卵母细胞纺锤体异常率为67.78%(61/90),对照组：18.35%(20/109)；X2=49.90,P<0.001,处理组显著高于对照组。CMPD1处理组染色体排列异常为63.33%(57/90),对照组异常率为21.10%(23/109),X2=36.56,P<0.001,处理组显著高于对照组。其异常主要表现为卵母细胞的染色体不规则略呈环形排列包绕着纺锤体；卵母细胞染色体滞后排列或者不规则散乱的染色体包裹这缩小的或者塌陷的纺锤体；一些染色体呈球状包绕着塌陷的纺锤体,表现为单极纺锤体。P-MK2信号从异常的纺锤体上消失或者减少。
6. MK2抗体注射对猪卵母细胞纺锤体组装、染色体排列和极体排放的影响对照组异常纺锤体率仅为21.0%(42/200),而抗体注射组,大量的纺锤体出现明显异常,呈现塌陷,缩小,球形或不规则散乱的染色体,纺锤体异常率高达66.9%(79/118),明显高于对照组(X2=66.47,P<0.001)；抗体注射组染色体错排率为70.34%(83/118),显著高于对照组(23.50%(47/200),X2=67.37,P<0.001)。抗体注射破坏MK2功能后显著影响了极体排出,抗体注射组第一极体排出率显著低于对照注射组(36.39%(31/124)&43.35(88/203)；X2=11.197,P<0.001)。
小结
1. MK2可以促进FSH诱导的猪卵母细胞卵丘的扩展。我们检测到卵丘上有MK2的表达,用MK2的抑制剂处理发现其显著抑制了猪卵丘的扩展,而且呈剂量依赖性,这一结果提示MK2可以促进FSH诱导的猪卵母细胞卵丘的扩展,这一作用可能通过颗粒细胞上的MK2发挥。
2. MK2调控猪卵母细胞减数分裂成熟。MK2抑制剂CMPD1显著抑制了COCs和裸卵的减数分裂进程,阻碍了第一极体的排放,但并没有影响猪卵自发的减数分裂恢复。
3. MK2参与了纺锤体组装和染色体分离。用MK2抑制剂处理猪裸卵,发现其显著破坏了卵母细胞的纺锤体组装和染色体排列；同时用p-MK2抗体注射裸卵,也检测到同样的结果,同时抗体注射还影响了猪卵第一极体的排放。故MK2参与减数分裂纺锤体组装和染色体排列。
全文小结
1.p38α能调节纺锤体和纺锤体极组装,参与染色体的分离。p-p38α定位于纺锤体两极并与在微管组装中心和胞浆星体上的γ-tubulin共定位；通过募集纺锤体极和MTOC上的γ-tubulin和Plk1参与微管成核；p38α通过调控动粒微管在纺锤体极的聚合以稳定纺锤体和纺锤体极。故p38α是小鼠卵母细胞无中心粒微管组装中心的一部分,也是微管负极相关蛋白。
2.p38α和Eg5在调节纺锤体的张力和长度时,相互拮抗,是一对反作用力p38α通过调控Eg5在微管上的分布来发挥调控纺锤体长度和大小的作用,但并不影响Dynein定位。p38α是一个重要的激酶,但是否通过磷酸化动力蛋白来调控纺锤体的张力还需进一步研究。
3.P38α通过影响小鼠卵母细胞BubR1这一重要的检验点蛋白来影响小鼠卵母细胞染色体分离,这是小鼠卵母细胞非整倍体发生的一个重要机制。
4. MK2可通过其颗粒细胞上的表达促进FSH诱导的猪卵丘的扩展,Mk2也参与了猪卵母细胞纺锤体组装和染色体分离,以及动粒和微管间的连接,阻碍了第一极体的排放,但并没有影响猪卵母细胞自发的减数分裂恢复。
5.p38α的功能有种属的差异,其表达在猪卵母细胞的胞浆,却表达在小鼠纺锤体的两极,而MK2在小鼠和猪卵母细胞上的定位相似。在小鼠卵母细胞中,p38α与MK2形成复合物,MK2是p38α的下游,p38α磷酸化其底物MK2,p38α通过调节小鼠卵母细胞减数分裂成熟中p38α-MK2信号通路参与纺锤体的组装,但在猪卵母细胞中MK2和p38α的关系不同于小鼠,其作用机制需进一步研究。
Objective
Abnormal germinal cell during genesis and maturation is the fundamental cause of infertility and birth-defect, which lead to failured fertilization and defective embryo development. The high incidence of chromosomally abnormal pregnancies in humans has been an intractable genetic puzzle for decades. Approximately30%of oocytes and9%of sperm may be chromosomally abnormal (aneuploid). Furthermore, the incidence is strongly affected by age. For women at the end of their reproductive lifespan, the risk of ovulating a chromosomally abnormal egg might be50%or higher. Approximately7-10%of clinically recognized pregnancies are chromosomally abnormal,60%of miscarriaged fetus are chromosomally abnormal;5-10%aborted fetus are chromosomally abnormal. High rate of aneuploid in human oocytes seriously affect human fertility and compromise the population qulity.
The quality of oocytes has the greatest influence on results of the monospermic fertilization, early development, and implantation of embryos. Therefore, the quality of oocytes can be a determining factor in the fertilization of oocytes, culture of high quality embryos, and treatment of the infertility. In humans, the oocyte plays a central role in providing RNAs and proteins that maintain genomic integrity of the zygote and cleavage-stage embryos until Day3when embryonic genome activation occurs60%embryos are anueploid, most of which can not be implanted. However, if the anueploid oocytes are implanted, the fetus will be miscarriaged and arrested development. High anueploid is the main reason of birth-defects, the low pregrant rate in ART and recurrent abortion, which were confirmed by preimplantation genetic detect in aged females and implantation failure.
The age of the mother is the main reason of the increased aneuploidy in birth defect. Germ cells go aging after fetus were born, only2-3%of the women at age of20years old have the oocytes with abnormal chromosomes, but at40, the risk increases to30-35%. The age increase the rate of anueploid in meiosis or mitosis of MI and MII oocytes. In recent30years, the age of the first birth increasing3.6year caused the high rate of abnormal chromosomes in ageing oocytes, which lead to the dramatic decline of fertility in female at35years old. Along with the mature of human assistant reproduction technology, high aneuploidy in oocytes is the main reason in the implantation failure. The reproduction in aged women is the giant challenge for assistant article technology. Some reports think that young women have show ed increasing recurrent spontaneous abortion, aneubploid oocytes and implantation failure after IVF. The phenomenon attribute to over-doses of FSH injection in control ovarian stimulation and progressively environmental pollution, which affected the recombination, synapsis development of oocytes in follicle pool at fetal period and oocytes susceptibility to aneuploidy.
Therefore, clarifing the mechanism of aneuploidy in female oocytes play an important role in reducing the generation of aneuploidy and approving the quality of oocytes. Chromosome segregation error lead to aneuploidy in oocytes, directly resulting in embryo development arrest or spontaneous abortion. The normal spindle assembly is the guarantee of correct chromosome segregation. The spindle pole assembly, the spindle form and length obviously affect the chromosome segregation. ensure correct spindle assembly and chromosome segregation. The proteins at spindle poles such as, protein kinase, dynein and microtuble-associated protein and spindle checkpoint protein interact with each other, which regulate the spindle assemble and chromosome assignment. The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK) and p38, all of which are highly conserved in all eukaryotic systems. MAPK (ERK) plays important roles in stabilizing and facilitating pole and chromosome separation. In addition, MEK, the upstream regulator of ERK participates in spindle assembly and chromosome alignment p38mitogen-activated protein kinase (p38MAPK) is one of the three major members of the MAPK family that regulates cellular responses including apoptosis, cell proliferation and immune response as well as cell growth, differentiation, and cytoskeletal rearrangements. A lot of research focus on somatic cell, but the mechanism of aneuploidy which is caused by abnormal chromosome segregation in oocytes. mitogen-activated protein kinase family (MAPK) pathway is the one of the most important signaling pathway, it is pivotal in many important biological progresses, such as cell proliferation, cell differentiation and cell cycle regulation. The p38protein is known to be phosphorylated by MKK3and MKK6for activation in response to cell stress and in turn it phosphorylates a number of substrates, including MAPKAP kinase2(MK2). P38a forms a stable heterodimer with MK2and mediates the phosphorylation and functions of MK2in vitro and in vivo. In the present study, we in the first explore the function of P38aMAPK on meiotic spindle organization and chromosome assignment in mouse oocytes, and understand the mechanism of aneuplord in the mammal oocyte.
Part one:p38a MAPK Is a MTOC-Associated Protein Regulating Spindle Assembly, Spindle Length and Accurate Chromosome Segregation during Mouse Oocyte Meiotic Maturation
The assembly of a functional bipolar spindle is critical for accurate chromosome segregation in mammalian oocytes. The size, shape and length of the spindle is relatively constant in one cell during spindle, which is regulated by motor proteins, protein kinase, microtubel-associated protein and pole-associated protein. The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK) and p38, all of which are highly conserved in all eukaryotic systems. p38mitogen-activated protein kinase (p38MAPK) is one of the three major members of the MAPK family that regulates cellular responses including apoptosis, cell proliferation and immune response as well as cell growth, differentiation, and cytoskeletal rearrangements. In the present study, we used the mouse oocytes as the model, investigating the roles of p38MAPK in the spindle assembly and spindle checkpoint activation, which enriches our knowledge about this cellular event in meiosis and provides the theoretical basis for the mechanisms forming the aneuploidy in mammalian oocytes.
Methods Immature oocytes were collected from ovaries of6-week-old female ICR mice. Firstly, we examined the expression and subcellular localization of this protein by immunoblotting analysis and immunofluorescence; next, we investigate the space localzation bwtween p38MAPK and MTOC in oocytes treated with Taxol。 Furthermore, we examined the phnotype in oocytes by special P38a inhibitor treatment and P38mophornilo injection. In addtion, we tested a possible interaction between endogenous p-p38a and p-MK2by co-immunoprecipitation with p-p38a antibody in mouse MI oocytes extracts and then performed immunoblot analysis with the anti-p-MK2antibody. Chromosome spreading was done for checking aneuploid in MII oocytes. At last, BubRl signal was detected in p38aMO-injected oocytes.
Results
1. Expression and subcellular localization of phospho-p38aMAPK (p-p38a) during mouse oocyte meiotic maturation The expression level of p-p38a remained stable from GV to MI stage, and then become reduced at the ATI and MII stages by Westernbloting. At the GV stage, p-p38a was localized in the germinal vesicle; after GVBD, multiple bright foci of p-p38a were detected near the individual chromosomes. By Pro-MI when chromosomes began to migrate to the equator of the spindle, p-p38a was gradually translocated to the spindle poles. Notably, p-p38a were specifically concentrated at the spindle poles at the MI stage. No evident signals of p-p38a were labeled in anaphase/telophase, however, p-p38a reappeared at the spindle poles in oocytes at the MII stage.
2.p-p38a colocalizes with y-tubulin at cytoplasmic MTOCs and spindle poles in mouse oocytes. p-p38a was colocalized with y-tubulin at the spindle poles and cytoplasmic MTOCs in oocytes at MI and MII stages. Taxol was employed for oocyte treatment. In this case, p-p38a was specifically colocalized with y-tubulin at the poles of the abnormal spindles, the center of MTOCs, and at the center of the cytoplasmic asters in oocytes at Pro-MI, MI and MII stages. Specifically, p-p38a was strictly colocalized with a broad "C"-shaped y-tubulin configuration unattached at the poles.
3.Depletion of p38α resulted in abnormal spindles and misaligned chromosomes To explore the roles of p38α, we employed a morpholino-based gene-silencing approach to deplete p38a in oocytes. Using a specific p38a-targeting morpholino (termed p38a-MO), we successful depleted most of the endogenous p-p38a protein in oocytes. P-p38a depletion did not affect meiotic cell cycle progression and rates of polar body extrusion. Down-regulation of p-p38a resulted in significant defects in spindle formation and chromosomes alignment. Aberrant spindle organization included elongated spindles and various defects in poles including multipolar spindles and disintegrated spindle poles. The rate of abnormal spindle formation in the p38aMO-injected group was74.4%(128/172), which was considerably higher than that of the control MO-injected group21.3%(40/188), x2=101.919, P<0.001. P38α-depleted oocytes displayed severe defects in chromosome alignment, showing lagging chromosomes and irregularly scattered chromosomes. The incidence of misaligned chromosomes in the p38aMO-injected group was up to75%(129/172), much higher than that in the control group (10.10%(19/188),(x2=156.241, P<0.001).
4. P38a depletion may compromise meiotic spindle organization and chromosome alignment via the p38α/MK2signaling pathway. Firstly, we tested a possible interaction between endogenous p-p38a and p-MK2by coimmunoprecipitation. p-MK2was clearly decreased in the p38a-depleted oocytes compared with the control group, indicating that p38a could phosphorylate and activate MK2. Subsequently, we examined the p-MK2localization after p38a depletion and demonstrated that p-MK2was dissociated from the spindle poles and chromosomes, and were scattered in the cytoplasm in the p38a-depleted oocytes.
5. p38a is required for recruitment of y-tubulin to MTOCs and stabilization of spindle bipolarity. In p38a-depleted MI oocytes, spindle organization was disrupted in over70%of oocytes (n=114). The typical defects in spindle assembly included aberrant poles (58.2%, n=114), with striking additional small poles around spindles or near the main poles. Significant γ-tubulin signals were observed in the cytoplasm in p38a-depleted MI oocytes. Moreover, MTOC asters labeled with y-tubulin loosely dispersed within the abnormal spindles or in the cytoplasm in MI oocytes, which indicates that activation of p38a is required for recruitment of cytoplasmic MTOCs to spindle poles. The oocytes with abnormal spindles displayed multiple bright foci loosely dispersed at the defective spindle poles. When treated with cold, microtubule-nucleating components became dissociated from the spindle ends in the p38a-depleted oocytes. Additionally, the minus ends of kinetochore microtubules were not tethered at the spindle poles. p38a was colocalized with Plkl at MTOCs and spindle poles in oocytes treated with taxol and in control oocytes. In p38a-depleted oocytes, Plkl was either completely dissociated from the spindle poles and chromosomes or Plkl signals became faint.
6. P38a and Eg5play opposite roles in determining spindle tension and length P38a-depleted oocytes displayed elongated spindles and increased tension between homologous kinetochores in pole-defocused spindles. The distance between homologous kinetochores in pole-defocused MI spindles of p38a-depleted oocytes (2.88μm±0.05, n=10spindles,64kinetochore pairs) was significantly increased compared to that of control oocytes (0.99μm,±0.04, n=8spindles,52kinetochore pairs) F=21.567, P<0.001, indicating that the tension between homologous kinetochores in pole-defocused spindles was notably increased. To quantify spindle length, we measured the spindle length as shown in the schematic. The spindles were50%longer in p38aMO-injected oocytes than those of control MO-injected oocytes (23.3±3.02μm). Interesting, spindle elongation was observed in Eg5-overexpressed oocytes (37.80±2.09μm). In contrast, monopolar spindles were observed in Eg5knocked-down oocytes. We further injected oocytes with both p38αMO and Eg5MO. As expected, in p38αMO and Eg5MO co-injected oocytes (25.3±2.72μm), bipolar spindle morphology and length were rescued.
7. P38a depletion affected the distribution of Eg5along microtubules in abnormal spindles. The results indicated that Eg5or dynein depletion did not affect the localization of p38a, and p38a was still localized at spindle poles. However, p38a depletion significantly affected the distribution of Eg5along the spindles. Eg5was localized at microtubules and concentrated at spindle poles in control MO injected oocytes. In Eg5-overexpressed oocytes, Eg5was localized to microtubules but not concentrated at the poles. However, in p38α-depleted oocytes, the expression of Eg5was decreased at poles, but increased on the spindle microtubules, notably at the midzone of the spindle. In some spindles, Eg5signal was only detected in half of the whole spindle.
8. P38a depletion inactivated SAC and increased frequencies of aneuploidy. We observed that the ratio of polar body extrusion (PBE) in p38a-depleted oocytes (78.2%, n=234) was not different from that of the control group (79.8%, n=196). However, p38a-delepted MI and MII oocytes showed obviously misaligned chromosomes. Thus, chromosome spreads were performed in p38a-depleted MII oocytes. Our results showed that p38a-depleted MII oocytes typically displayed incorrect numbers (more or less than20) of univalents. The frequency of aneuploidy of p38a-depleted MII oocytes42.4%(14/33) was significantly higher than that in control oocytes (4.8%(1/21))x2=9.07, P=0.003, P<0.05. We observed that BubRl was enriched at numerous kinetochores in control MO injected oocytes at the prometaphase stage. However, no BubRl signal was detected in p38aMO-injected oocytes.
Summary
1. P38a is an important component of MTOC, which regulates spindle assembly. P38a specifically colocalized with y-tubulin and Plkl at the center of MTOCs and spindle poles. Knockdown of p38a lead to the disperse of y-tubulin and Plkl at spindle pole, which support that p38a regulate spindle and microtuble nuclear and recruit y-tubulin to MTOC. Depletion of p38a by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2dephosphorylation.
2. P38a stablized the spindle and spindle pole in mouse oocyte. Depletion of p38a led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased y-tubulin and Plkl at MTOCs, which indicate that P38a connected the pole microtuble and MTOC with the negative of microtuble. The unclustered pole microtuble and kinetochore microtuble can not be assembled into normal spindle pole after P38a, which indicate that P38a is an important component of pole-associated proteins.
3. p38a antagonize the Eg5by indirectly producing a counteracting force, which regulate the tension and length of the spindle. Depleted-P38a led to inccreased spindle length and microtubel tension. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation, and its morpholino injection almost completely rescued spindle elongation caused by p38a depletion, the presence of an antagonistic relationship between Eg5and p38a in mouse oocytes. Given that Eg5generates an outward force by sliding antiparallel microtubules, we considered the possibility that p38a may affect other minus proteins, which generate an inward force and partially antagonized Eg5. Thus, Eg5could be responsible for spindle elongation with increased tension in the absence of p38a. P38a depletion led to imbalance in force between spindle poles, which may be explained the abnormal poles in the absence of p38a.
4. p38a play an important pole in the formation of anueploid oocytes by regulating the spindle assemble checkpoint protein on kinetochores in mouse oocytes. Impairment of p38a disturbed the localization of BubRl at the kinetochores of chromosomes and compromised the SAC, which caused misaligned chromosome and subsequent aneuploidy in mouse oocyte meiosis.
Part two MK2Regulates Spindle Assembly and Accurate Chromosome Segregation during porcine Oocyte Meiotic Maturation
Methods
Porcine COCs were collected for culture in vitro. Firstly, the cumulus cell expansion and meiotic maturation of COCs were checked after the specific MK2inhibitor CMPD1treatment. Next, the meiotic maturation of DOs were investigated after CMPD1treatment. MK2were accurately located in spindle and chromosome、of porcine oocytes by immunofluorescence. To further clarify the correlation among MK2, MTOC, Crest and Plkl, taxol was employed for oocyte treatment. Furthermore, the spindle formation and chromosome alignment were explored after depletion of MK2by CMPD1treatment and MK2antibody injection.
Results
1. The effect of CMPD1on the cumulus expansion of porcine COCs After porcine COCs were cultured for24h in TMP-199with FSH,85.7%(281/328) of COCs in control were examined as cumulus expansion. High density CMPD1(30μmol/L) significantly inhibited the cumulus expansion (3.3%(8/242));12.5%(30/240)&24.39%(80/328) were expanded in the presence of20μmol/L and10μmol/L(χ2=546.22, P<0.001), which indicate that CMPD1inhibits porcine cumulus expansion dependly on the concentration of CMPD1.
2. Evaluating the effect of CMPD1on porcine COCs meiotic maturation. CMPD1dose not affect the meiotic recover of porcine oocyte, however, CMPD1affects the extrusion of the first polar body(pbl). After porcine COCs were cultured for42h in TMP-199with FSH, the rate of pbl extrusion in porcine oocytes in30,20,10μmol/L showed as0%;6.25%(11/176)&37.9%(50/132), which significantly decreased in comparison with the control (63.16%(60/95),χ2=178.17, P<0.001). Thus, CMPD1inhibit the pbl extrusion in porcine oocytes dependly on the concentrtion of CMPD1.
3. Evaluating the effect of CMPD1on meiotic maturation of porcine denude oocytes. CMPD1dose not affect the the meiotic recover of porcine oocyte but the pb1extrusion. The rate of pb1extrusion of porcine oocytes in30,20,10μmol/L showed as0%(0/136);5.41%(10/185)&19.48%(30/154), which significantly decreased in comparison with the control54.46%(55/101),χ2=150.055, P<0.001. Moreover, the rate of pbl extrusion in DO treated with10μmol/L CMPD1siginificantly decreased compared with the control (54.46%(55/101)).
4. The subcellular location of p-MK2during porcine oocyte meiotic maturation. After germinal vesicle breakdown (GVBD), p-MK2was localized along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions. When oocytes progressed to MI, homologous chromosomes aligned at the equatorial plate, and p-MK2localized at the spindle plus ends and chromosomal axes. At anaphase I, the homologous chromosomes were segregated, and the p-MK2signals disappeared from chromosomes and localized to the equatorial region between the separating chromosomes. At MII, the p-MK2signals again translocated to the spindle plus ends and was located between Crest and sister chromatids. P-MK2was partly colocalized with Plkl on chromosome and microtubles or was located between Crest and sister chromatids. P-MK2was partly colocalized was y-tubulin on spindle.
5. MK2inhibitor CMPD1affect the spindle assembly and chromosome assignment in porcine oocytes. Down-regulation of MK2resulted in significant defects in spindle formation and chromosomes alignment. Aberrant spindle organization included collapsed spindle surrounded by dispersed chromsomes and irregularly scattered spindle. The rate of abnormal spindle formation in CMPD1treatment group was67.78%(61/90), which was considerably higher than that of the control group18.35%(20/109);χ2=49.90, P<0.001. P38a-depleted oocytes displayed severe defects in chromosome alignment, showing lagging chromosomes and irregularly scattered chromosomes. The incidence of misaligned chromosomes in CMPD1treatment group was up to63.33%(57/90), much higher than that in the control group21.10%(23/109),χ2=36.560, P<0.001.
6. MK2antibody injection disrupted spindle assemble and chromosome assignment in porcine oocytes. The rate of abnormal spindle formation in antibody-injected group was66.9%(79/118), which was considerably higher than that of the control group21.0%(42/200);(χ2=66.47, P<0.001); the incidence of misaligned chromosomes in antibody-injected group was up to70.34%(83/118), much higher than that in the control group_(23.5%(47/200χ2=67.37, P<0.001).
Summary
1. MK2play an important role in cumulus expansion of porcine oocytes induced by FSH through the MK2on the cumulus. MK2inhibitor CMPD1significantly inhibited the cumulus expansion of COCs.
2. MK2regulates spindle assemble and chromosome assignment in porcine oocytes. collapse spindles surrounded by the chromosomes is a specific phenotype, which indicates that MK2can connected microtuble with crest. The results from CMPD1treatment and antibody injection support the above views.
3. MK2regulates the procession of porcine oocytes during meiotic maturation. However, MK2dose not affect the spontaneous meiotic recovery in GV oocytes.
Conclusion
1. p38aMAPK regulate spindle, spindle pole organization and chromosome assignment. p-p38α was located at spindle pole and was colocalized with γ-tubulin at MTOC and cytoplasm aster; p38aMAPK can recuit Plk1and γ-tubulin to spindle pole and MTOC and promote the microtuble nucleation. p38aMAPK regulates kintochore microtuble polymerized and stablized the spindles and spindle poles. Thus, p38aMAPK is one component of MTOCs in mouse oocytes and also is a negative microtuble-associated protein.
2. p38a and Eg5are an antagonism force, which regulate spindle tension and length. p38a can regulate spindle tension and length through changing Eg5distribution at microtubule, but not affect the location of Dynein. p38a is an important kinase. It needs to be further investigated whether p38aMAPK regulates spindle tension through phosphrylating motor proteins.
3. p38a affects the spindle checkpoints BubR1in mouse oocytes and regulates chromosome assignment, which is an important mechanism about mouse aneuploid.
4. MK2promotes porcine comulus expandation through FSH induction on the cumulus. MK2also regulates spindle assemble, chromosome separation and connected the Crest with microtubles in porcine oocytes. MK2interupts the pbl extrusion, but not affect the spontanous meiotic recovery in porcine oocytes.
5. The location of p38a is charaterized with species distinction. p38a is localized at the cytoplasm of porcine oocytes, but at the spindle poles in mouse oocytes. However, the location of MK2in mouse is the same as in the porcine oocytes. In mouse oocytes, p38a is formed a compound with MK2and MK2is the downstream of p38aMAPK, which phosphrylates Mk2. p38a regulates spindles assemble and chromosome assignments through p38aMAPK-MK2pathway. However, the relatation between p38aMAPK and MK2in the porcine oocytes is different from the mouse oocytes. Thus the mechanism about MK2in porcine oocytes need to further be explored.
不孕不育和出生缺陷的根本原因是生殖细胞在发生和成熟过程中的异常造成受精和胚胎发育的失败或缺陷。近几十年来,人类妊娠中异常高发的非整倍体率二直是个顽固的遗传难题。据统计，人类大约有30%的卵子和9%的精子携带有不平衡的染色体；而且大龄女性排出的卵子发生染色体异常的风险大于50%；有7-10%的临床妊娠伴随染色体异常；自然流产胎儿中有60%为非整倍体所致,人工流产胎儿中5-10%为非整倍体。卵子高发的非整倍体严重影响了女性的生殖力。
卵子是生殖技术的基础,卵子的质量大大影响了受精、胚胎的早期发育和着床,因此卵子质量是不孕患者在辅助生殖中卵子受精,胚胎着床的保证。卵子也为合子和受精后前三天的胚胎提供RNA和蛋白,以营养和维持基因的稳定性,直至胚胎基因被激活’。有报道称辅助生殖中60%的人胚胎为非整倍体,大部分都不能着床,但是非整倍体胚胎一旦种植,就会发生早期发育停滞和流产。对大龄女性,反复着床失败、复发性流产和有非整倍体病史者行种植前的遗传学检查也证实非整倍体是出生缺陷,辅助生殖技术成功率低和复发性流产的主要原因之一。
母亲年龄是导致出生缺陷中非整倍体增加的主要病因。女性的生殖细胞一出生就意味着逐渐走向衰老,20±岁的年轻妇女排出的卵母细胞仅有2-3%发生染色体异常,而40岁时,这种风险增至30-35%。年龄增加了卵母细胞第一次和第二次减数分裂发生错误率。近30年来,女性第一次生育的年龄已经增加了3.6岁,导致了35岁后生育的女性明显增加,而大于35岁后生育的女性由于卵子染色体异常率高导致生育力明显下降。随着人类辅助生殖技术的迅速普及和成熟,由卵子非整倍体异常导致的着床失败所占比例逐渐增加,大龄妇女的生育是目前面临的一个挑战。而且有文献报道在辅助生殖体外受精后年轻女性发生复发性流产,反复非整倍体发生或种植失败也明显增加,这可能归因于超排卵中大剂量外源性的FSH应用和日益恶劣的环境污染,而且会影响胎儿期卵泡池中卵子的重组、联会和生长,增加成年后卵子对非整倍体的易感性,可能影响子代的配子质量2。
因此,研究女性卵子非整倍体的发病机制,减少非整倍体的发生,提高卵子质量是目前生殖领域最重要的课题之一。卵母细胞染色体不分离所致的非整倍体,直接导致胚胎停育或流产。正常的纺锤体组装是染色体正确分离的保障。其中纺锤体极的组装,纺锤体形状和长度也明显影响染色体的分离。只有通过蛋白激酶、磷酸化酶、动力蛋白、微管相关蛋白和纺锤体检验点蛋白等相互作用才能保证准确的纺锤体组装和染色体分离。目前对体细胞非整倍体发生机制的研究已非常深入,但是卵母细胞染色体分离异常所致的非整倍体发病机制不清。丝裂源活化蛋白激酶超家族(MAPK)信号通路是细胞内最重要的信号通路之一,它在许多重要的生物活动中起到至关重要的作用,如调控细胞增殖、分化以及细胞周期等。丝裂源活化蛋白激酶超家族是由经典的细胞外调节激酶(ERK), c-Jun或应激激活蛋白激酶(JNK)和p38组成,这些家族中的所有成员在真核细胞系中高度保守。ERK参加纺锤体组装和染色体的分离；另外作为MAPK的上游调控激酶MEK,它可能作为中心体蛋白,在微管组装,纺锤体极的聚合以及胞质不均等分裂中起作用。P38MAPK是MAPK家族中三个主要成员之一,它在调节细胞的生长分化；细胞的凋亡,增生以及细胞免疫反应和细胞骨架的重排中起到重要的作用。在体细胞中,p38a与MK2形成一个稳定的异源二聚体,在体内外磷酸化MK2并调节其功能。P38MAPK也参与前间期、G2/M细胞周期检验点功能,但p38MAPK是否是个有丝分裂的纺锤体组装检验点蛋白一直存在争议。而且本研究组前期已对MK2在小鼠卵母细胞减数分裂中的作用进行了探讨,发现其在纺锤体组装和同源染色体的分离中起到重要作用。在本研究中,我们首次对p38MAPK在小鼠卵母细胞减数分裂中的作用以及MK2在猪卵母细胞中的作用进行研究,以探讨其在哺乳动物卵母细胞非整倍体发生机制中的作用,为提高卵子质量、减少卵子和胚胎的非整倍性发生、辅助生殖技术的完善、生殖疾病防治和出生缺陷的减少奠定理论基础。
第一部分：P38αMAPK在小鼠卵母细胞减数分裂纺锤体组装和染色体分离中的作用
正常的纺锤体组装是确保染色体分离的重要前提。在纺锤体组装中,一定的细胞,其纺锤体的大小、形状和长度都相对固定,并受到动力蛋白、蛋白激酶和微管相关蛋白和极蛋白的调控。P38MAPK是MAPK家族中三个主要成员之一,在体细胞中已有研究认为其在细胞骨架的重排中起到重要作用。因减数分裂和有丝分裂的发生机制存在差异,减数分裂更为复杂,故各种蛋白在体细胞和卵母细胞中的作用不相同,本研究通过对P38MAPK在小鼠卵母细胞减数分裂中的作用探讨,丰富了我们对减数分裂这一重要细胞事件的了解,并为哺乳动物卵母细胞非整倍体的形成机制提供理论依据。
研究方法
取6周龄的ICR雌性小鼠的GV期卵母细胞在体外成熟培养,首先我们用免疫荧光对p38aMAPK进行准确定位；用免疫印迹检测p38aMAPK在小鼠各期卵母细胞中的蛋白表达水平；接着用免疫荧光检测Taxol处理后的卵母细胞p38aMAPK与微管组织中心及其相关蛋白的空间定位关系；进一步我们用p38aMAPK抑制剂、p38aMAPK mophornilo寡居核苷酸(p38a-MO)注射卵母细胞,抑制p38aMAPK的磷酸化或蛋白表达后研究其表型；同时我们用免疫共沉淀的方法检测了p38αMAPK和MK2的相互作用；接着我们用染色体铺片的方法证实MⅡ卵非整倍体的发生；最后用免疫荧光检测p38αMAPK功能破坏后对前中期卵母细胞中检验点蛋白BubRl的影响来探讨p38αMAPK对检验点蛋白的调控作用。
结果
1.磷酸化的p38αMAPK (p-p38α)在小鼠卵母细胞减数分裂成熟中的蛋白表达及亚细胞定位免疫印迹结果显示从生发泡期(GV)至中期(MI),p-p38a的蛋白表达量一直保持稳定,自后期到MⅡ期,蛋白表达量逐渐减少。p-p38a亚细胞定位：生化泡破裂期(GVBD)定位在染色体附近；前中期(Pre-MI),开始向纺锤体两极移动；MI期,p-p38a定位在纺锤体两极；中后期,没有检测到明显的p-p38a的信号；MⅡ期,p-p38a再次回到纺锤体极上。
2.p-p38a在小鼠卵母细胞减数分裂成熟中与中心体蛋白γ-tubulin定位关系在正常卵母细胞中生发泡破裂后,p-p38a在整个减数分裂成熟过程中都与γ-tubulin共定位；使用taxol微管聚合剂处理卵母细胞后,在前中期,中期和MⅡ期的异常纺锤体极,微管组装中心(MTOC)和胞浆星体的中心都能检测到p-p38a的信号,而且与γ-tubulin严格共定位。
3.评估破坏p-p38a功能后小鼠卵母细胞减数分裂中纺锤体组装和染色体排列p38a特异性的抑制剂SD203580处理GV卵后,影响了小鼠卵母细胞减数分裂中纺锤体组装和染色体排列。抑制剂组纺锤体异常率为70.5%(103/146),对照组18.54%(28/151),抑制剂组显著高对照组(χ2=81.430,P<0.001)；抑制剂组染色体错误排列率为61.64%(90/146),显著高于对照组13.25%(20/151),(χ2=74.563,P<0.001)。p38α-MO注射后影响了小鼠卵母细胞减数分裂中纺锤体组装和染色体排列,p38α-MO注射组中染色体异常率75.0%(129/172),与对照MO注射组(10.10%(19/188))比,染色体错排率显著升高(χ2=156.241,P<0.001)；此外,p38α-MO注射组中纺锤体异常率为74.4%(128/172),对照MO注射组21.3%(40/188),χ2=101.919,P<0.001,p38α-MO注射组显著高于对照组。
4.p38α与MK2在小鼠卵母细胞中的相互关系免疫共沉淀证实内源性的p38α与MK2能相互作用；p38α-MO注射后的卵母细胞中p-MK2蛋白的表达水平下降说明p38α能磷酸化MK2；p38α基因干扰后影响了p-MK2的定位,p-MK2从纺锤极和染色体上脱落,散落在卵的胞浆中,这一结果证实p38α能影响MK2的定位。
5.p38α对MI卵母细胞纺锤体极组装的影响MI卵母细胞纺锤体组装中典型的异常包括：极紊乱是纺锤体异常最突出的表现之一(实验组：57.89%&对照组：7.94%)；其它主要表现为一些单极(实验组：14.91%&对照组：3.97%)或另外的小极围绕着纺锤体或在极的附近,对照组与p38a-MO注射组比,Z=98.095,df=3,P<0.001(双侧),并可见γ-tubulin和标有γ-tubulin的MTOC星体分散在异常的纺锤体和MI卵的胞浆中。对p38a干扰后的卵母细胞行冷处理破坏非动粒微管后.,动粒微管完全不能聚合在纺锤体极韵两端。正常及Taxol处理后的卵母细胞,p-p38a与Plk1在MTOC和纺锤体极上严格共定位,p38a干扰后,Plk1完全从纺锤体极和染色体上脱落。
6.p38a或Eg5干扰及Eg5过表达后对纺锤体张力和长度的影响p38a干扰后,通过检测同源染色体动粒间的距离来评估其张力：p38a-MO注射组动粒间的距离为2.88±0.05-n,显著长于对照组(0.99±0.041μm),F=21.567,P<0.001,t=-47.985。纺锤体长度：p38α-MO注射组(39.5+3.37μm)显著长于对照组(23.3±3.02μm)(P<0.001)；Eg5过表达组纺锤体明显拉长(37.80±2.09μm),显著长于对照组(P<0.001)；Eg5过表达后与p38a-MO注射组纺锤体长度比,无统计学差异(P=0.163,P>0.05)；而Eg5功能缺失的卵却表现为单极纺锤体；同一个卵中同时注射p38a-MO和Eg5-MO后,双极纺锤体的形态和长度部分恢复(25.3±2.72μm),与对照组比无统计学差异(P=0.093,P>0.05),但形态不稳定。
7.p38α干扰后对Eg5和Dynein在纺锤体上分布的影响破坏Eg5和Dynein功能后,p38α在纺锤体上的定位无改变；正常卵母细胞：Eg5定位在微管和纺锤体极上；过表达Eg5后,Eg5均匀的定位在纺锤体的微管上,双极无表达；p38α基因干扰后：Eg5在极上的表达减少,而在纺锤体微管上的表达增加,尤其在纺锤体中板上的表达显著增加。
8.p38α功能破坏后,对MⅡ卵染色体排列和非整倍体率的评估以及对检验点蛋白BubR1的影响p38a缺失后并没有影响第一极体的排出(p38MO：78.2%,＆对照组：79.8%,n=196；χ2=0.123,P=0.730,P>0.05)；MⅡ卵表现明显的染色体排列异常,p38a-MO注射组MⅡ卵染色体异常率为65.70%(71/108),对照组MⅡ卵染色体异常率为7.9%(10/126),p38a-MO注射组染色体排列异常率显著高于对照组,χ2=85.850,P<0.001；MⅡ卵非整倍体率统计结果：p38a-MO注射组非整倍体率为42.4%(14/33),而对照组为4.8%(1/21)；p38a-MO组非整倍体率显著高于对照组,χ2=9.07,P=0.003,P<0.05。在注射p38α-MO后前中期卵母细胞中,大部分定位在卵母细胞上的BubR1从动粒上消失。
小结
1.p38α是小鼠卵母细胞无中心粒微管组装中心的一部分,能调节纺锤体的组装和染色体的排列p-p38α定位于纺锤体两极并与在微管组装中心和胞浆星体上的γ-tubulin和Plkl共定位,说明其是MTOC的一部分,参加了微管的组装和成核；p38α缺失导致了γ-tubulin和Plkl从纺锤体极上脱落,支持p38α参与微管成核,并能募集γ-tubulin到MTOC和参与纺锤体组装；p38α功能破坏导致了纺锤体形状异常和染色体排列紊乱,进一步支持p38α是减数分裂纺锤体组装中必不可少的蛋白激酶。p38α与MK2在小鼠卵母细胞中形成复合物,MK2是p38α的下游,p38α磷酸化其底物MK2,p38α通过小鼠卵母细胞减数分裂成熟中p38α-MK2信号通路参与纺锤体的组装和染色体的分离。
2.p38α能稳定纺锤体和纺锤体极。p38α功能缺失导致纺锤体组装异常,表现为多极,单极和极松散不粘合,有γ-tubulin信号的完整环形极从纺锤体上脱落,这一现象表明p38α能将极微管和无中心体的微管组装中心与微管的负极偶联；冷处理p38α-MO注射后MI卵破坏非动粒微管后,能更清晰的观察到动粒微管的负极末端不能粘合；这些结果都表明p38α能稳定纺锤体和纺锤体极。
3.p38α和Eg5在调节纺锤体的张力和长度时,相互拮抗,是一对反作用力量p38α功能的缺失导致了纺锤体的拉长和微管张力的增加,Eg5缺失导致了单极纺锤体,而过表达Eg5导致了纺锤体的拉长,和p38α功能缺失后的结果一致；P38α功能的缺失破坏了Eg5这一重要驱动蛋白在微管上的分布,故P38α和Eg5相互拮抗,通过影响驱动蛋白Eg5在纺锤体上的定位发挥调控纺锤体长度和大小的作用。
4.P38α调节小鼠卵母细胞纺锤体检验点。P38α功能缺失阻碍了BubR1在染色体动粒上的定位而损害了纺锤体检验点的功能,这是小鼠卵母细胞减数分裂中引起染色体排列异常且非整倍体率增加的重要原因。
第二部分：MK2在猪卵母细胞减数分裂纺锤体组装和染色体分离中的作用
正常的纺锤体组装是确保染色体分离的重要前提。在体细胞和小鼠卵母细胞中P38α和MK2形成一个复合体,P38α磷酸化MK2并影响MK2的定位,在小鼠卵母细胞中P38α通过P38α-MK2信号通路作用于其底物MK2调控纺锤体的组装和染色体的分离。我们研究组前期发现小鼠中MK2定位在纺锤体和染色体臂间,也在纺锤体组装和同源染色体的分离中起到重要作用。可是P38α在猪卵母细胞中不同于小鼠卵母细胞中的定位,其定位在猪卵母细胞的胞浆中,同是哺乳动物,却发现其有表达差异,这一结果提示我们进一步探讨MK2在猪卵母细胞减数分裂中的作用,并比较其和小鼠卵母细胞中的差异,以进一步完善MK2在哺乳动物卵母细胞减数分裂中的作用机制。
研究方法
取猪卵丘-卵母细胞复合物(COCs)在体外培养成熟,首先我们用MK2的抑制剂CMPD1处理COCs观察卵丘扩展以及对减数分裂进程的影响；接着用CMPD1处理裸卵,以观察MK2抑制剂CMPD1对裸卵减数分裂成熟的影响。用免疫荧光对MK2在猪卵母细胞的纺锤体和染色体上进行准确定位；其次用Taxol处理卵母细胞后用免疫荧光检测其与微管组装中心和Plk1、Crest和γ-tubulin的关系：再次我们用MK2抑制剂和p-MK2抗体分别注射卵母细胞,破坏MK2的磷酸化及其功能后研究其表型；最后用免疫荧光检测MK2功能破坏对Plkl定位的影响。
结果
1.MK2抑制剂CMPD1对猪卵丘-卵母细胞复合物卵丘扩展的影响猪卵丘细胞复合物在含有FSH的培养基中培养24h后,对照组卵丘扩展率为85.7%(281/328),然而高浓度的CMPD1(30μmol/L)几乎完全抑制了由FSH诱导的卵丘扩展(3.3%(8/242))；20μmol/L和10μmol/L组卵丘扩展率分别为：12.5%(30/240)＆24.39%(80/328)；χ2=546.22,P<0.001；故30,20,10和0μmol/L各处理组间有显著的差异,CMPD1呈剂量依赖性的抑制FSH诱导的卵丘扩展。
2.评价MK2抑制剂CMPD1对猪卵丘-卵母细胞复合物减数分裂成熟的影响MK2抑制剂CMPD1不影响猪卵母细胞自发的减数分裂恢复,但却阻碍了卵母细胞极体的排放。猪卵丘-卵母细胞复合物在含有FSH的培养基中培养42h,63.16%(60/95)的卵母细胞排放了第一极体,然而高浓度的CMPD1(30μmol/L)完全抑制了猪卵母细胞第一极体的排放；20pmol/L和10μmol/L的CMPD1的极体排放率为(6.25%(11/176)＆37.9%(50/132),χ2=179.045；P<0.001,故CMPD1呈剂量依赖性的抑制卵母细胞第一极体的排出。
3. MK2抑制剂CMPD1对猪裸卵减数分裂成熟的影响不同浓度CMPD1处理均未影响猪裸卵自发性减数分裂恢复；对照组裸卵极体排放率为54.46%(55/101),而高浓度的CMPD1(30μmol/L)处理完全抑制了猪卵母细胞第一极体的排放,极体排放率为0%(0/136);20μmol/L和10μmol/L CMPD1处理后第一极体排放率分别为5.41%(10/185)&19.48%(30/154)；χ2=150.055,P<0.001。10μmol/L CMPD1对猪裸卵(19.5%(30/154))第一极体排放的影响显著大于对COCs(37.9%(50/132)),χ2=11.94,P<0.001。
4.检测猪卵母细胞中MK2的亚细胞定位前中期和中期p-MK2沿着染色体臂定位于臂轴间隙和着丝粒区域,且与微管部分共定位,但是表达的面积小于α-tubulin;MⅡ期,p-MK2呈点状定位在动粒与姐妹染色体间,也就是姐妹染色体的着丝粒区。p-MK2与Plk1在微管上部分共定位,Plk1定位在染色体的动粒上,而p-MK2定位在染色体与Plk1之间。p-MK2与γ-tubulin在纺锤体上部分共定位。
5.观察MK2抑制剂CMPD1对猪卵母细胞纺锤体组装和染色体排列的影响CMPD1处理组卵母细胞纺锤体异常率为67.78%(61/90),对照组：18.35%(20/109)；X2=49.90,P<0.001,处理组显著高于对照组。CMPD1处理组染色体排列异常为63.33%(57/90),对照组异常率为21.10%(23/109),X2=36.56,P<0.001,处理组显著高于对照组。其异常主要表现为卵母细胞的染色体不规则略呈环形排列包绕着纺锤体；卵母细胞染色体滞后排列或者不规则散乱的染色体包裹这缩小的或者塌陷的纺锤体；一些染色体呈球状包绕着塌陷的纺锤体,表现为单极纺锤体。P-MK2信号从异常的纺锤体上消失或者减少。
6. MK2抗体注射对猪卵母细胞纺锤体组装、染色体排列和极体排放的影响对照组异常纺锤体率仅为21.0%(42/200),而抗体注射组,大量的纺锤体出现明显异常,呈现塌陷,缩小,球形或不规则散乱的染色体,纺锤体异常率高达66.9%(79/118),明显高于对照组(X2=66.47,P<0.001)；抗体注射组染色体错排率为70.34%(83/118),显著高于对照组(23.50%(47/200),X2=67.37,P<0.001)。抗体注射破坏MK2功能后显著影响了极体排出,抗体注射组第一极体排出率显著低于对照注射组(36.39%(31/124)&43.35(88/203)；X2=11.197,P<0.001)。
小结
1. MK2可以促进FSH诱导的猪卵母细胞卵丘的扩展。我们检测到卵丘上有MK2的表达,用MK2的抑制剂处理发现其显著抑制了猪卵丘的扩展,而且呈剂量依赖性,这一结果提示MK2可以促进FSH诱导的猪卵母细胞卵丘的扩展,这一作用可能通过颗粒细胞上的MK2发挥。
2. MK2调控猪卵母细胞减数分裂成熟。MK2抑制剂CMPD1显著抑制了COCs和裸卵的减数分裂进程,阻碍了第一极体的排放,但并没有影响猪卵自发的减数分裂恢复。
3. MK2参与了纺锤体组装和染色体分离。用MK2抑制剂处理猪裸卵,发现其显著破坏了卵母细胞的纺锤体组装和染色体排列；同时用p-MK2抗体注射裸卵,也检测到同样的结果,同时抗体注射还影响了猪卵第一极体的排放。故MK2参与减数分裂纺锤体组装和染色体排列。
全文小结
1.p38α能调节纺锤体和纺锤体极组装,参与染色体的分离。p-p38α定位于纺锤体两极并与在微管组装中心和胞浆星体上的γ-tubulin共定位；通过募集纺锤体极和MTOC上的γ-tubulin和Plk1参与微管成核；p38α通过调控动粒微管在纺锤体极的聚合以稳定纺锤体和纺锤体极。故p38α是小鼠卵母细胞无中心粒微管组装中心的一部分,也是微管负极相关蛋白。
2.p38α和Eg5在调节纺锤体的张力和长度时,相互拮抗,是一对反作用力p38α通过调控Eg5在微管上的分布来发挥调控纺锤体长度和大小的作用,但并不影响Dynein定位。p38α是一个重要的激酶,但是否通过磷酸化动力蛋白来调控纺锤体的张力还需进一步研究。
3.P38α通过影响小鼠卵母细胞BubR1这一重要的检验点蛋白来影响小鼠卵母细胞染色体分离,这是小鼠卵母细胞非整倍体发生的一个重要机制。
4. MK2可通过其颗粒细胞上的表达促进FSH诱导的猪卵丘的扩展,Mk2也参与了猪卵母细胞纺锤体组装和染色体分离,以及动粒和微管间的连接,阻碍了第一极体的排放,但并没有影响猪卵母细胞自发的减数分裂恢复。
5.p38α的功能有种属的差异,其表达在猪卵母细胞的胞浆,却表达在小鼠纺锤体的两极,而MK2在小鼠和猪卵母细胞上的定位相似。在小鼠卵母细胞中,p38α与MK2形成复合物,MK2是p38α的下游,p38α磷酸化其底物MK2,p38α通过调节小鼠卵母细胞减数分裂成熟中p38α-MK2信号通路参与纺锤体的组装,但在猪卵母细胞中MK2和p38α的关系不同于小鼠,其作用机制需进一步研究。
Objective
Abnormal germinal cell during genesis and maturation is the fundamental cause of infertility and birth-defect, which lead to failured fertilization and defective embryo development. The high incidence of chromosomally abnormal pregnancies in humans has been an intractable genetic puzzle for decades. Approximately30%of oocytes and9%of sperm may be chromosomally abnormal (aneuploid). Furthermore, the incidence is strongly affected by age. For women at the end of their reproductive lifespan, the risk of ovulating a chromosomally abnormal egg might be50%or higher. Approximately7-10%of clinically recognized pregnancies are chromosomally abnormal,60%of miscarriaged fetus are chromosomally abnormal;5-10%aborted fetus are chromosomally abnormal. High rate of aneuploid in human oocytes seriously affect human fertility and compromise the population qulity.
The quality of oocytes has the greatest influence on results of the monospermic fertilization, early development, and implantation of embryos. Therefore, the quality of oocytes can be a determining factor in the fertilization of oocytes, culture of high quality embryos, and treatment of the infertility. In humans, the oocyte plays a central role in providing RNAs and proteins that maintain genomic integrity of the zygote and cleavage-stage embryos until Day3when embryonic genome activation occurs60%embryos are anueploid, most of which can not be implanted. However, if the anueploid oocytes are implanted, the fetus will be miscarriaged and arrested development. High anueploid is the main reason of birth-defects, the low pregrant rate in ART and recurrent abortion, which were confirmed by preimplantation genetic detect in aged females and implantation failure.
The age of the mother is the main reason of the increased aneuploidy in birth defect. Germ cells go aging after fetus were born, only2-3%of the women at age of20years old have the oocytes with abnormal chromosomes, but at40, the risk increases to30-35%. The age increase the rate of anueploid in meiosis or mitosis of MI and MII oocytes. In recent30years, the age of the first birth increasing3.6year caused the high rate of abnormal chromosomes in ageing oocytes, which lead to the dramatic decline of fertility in female at35years old. Along with the mature of human assistant reproduction technology, high aneuploidy in oocytes is the main reason in the implantation failure. The reproduction in aged women is the giant challenge for assistant article technology. Some reports think that young women have show ed increasing recurrent spontaneous abortion, aneubploid oocytes and implantation failure after IVF. The phenomenon attribute to over-doses of FSH injection in control ovarian stimulation and progressively environmental pollution, which affected the recombination, synapsis development of oocytes in follicle pool at fetal period and oocytes susceptibility to aneuploidy.
Therefore, clarifing the mechanism of aneuploidy in female oocytes play an important role in reducing the generation of aneuploidy and approving the quality of oocytes. Chromosome segregation error lead to aneuploidy in oocytes, directly resulting in embryo development arrest or spontaneous abortion. The normal spindle assembly is the guarantee of correct chromosome segregation. The spindle pole assembly, the spindle form and length obviously affect the chromosome segregation. ensure correct spindle assembly and chromosome segregation. The proteins at spindle poles such as, protein kinase, dynein and microtuble-associated protein and spindle checkpoint protein interact with each other, which regulate the spindle assemble and chromosome assignment. The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK) and p38, all of which are highly conserved in all eukaryotic systems. MAPK (ERK) plays important roles in stabilizing and facilitating pole and chromosome separation. In addition, MEK, the upstream regulator of ERK participates in spindle assembly and chromosome alignment p38mitogen-activated protein kinase (p38MAPK) is one of the three major members of the MAPK family that regulates cellular responses including apoptosis, cell proliferation and immune response as well as cell growth, differentiation, and cytoskeletal rearrangements. A lot of research focus on somatic cell, but the mechanism of aneuploidy which is caused by abnormal chromosome segregation in oocytes. mitogen-activated protein kinase family (MAPK) pathway is the one of the most important signaling pathway, it is pivotal in many important biological progresses, such as cell proliferation, cell differentiation and cell cycle regulation. The p38protein is known to be phosphorylated by MKK3and MKK6for activation in response to cell stress and in turn it phosphorylates a number of substrates, including MAPKAP kinase2(MK2). P38a forms a stable heterodimer with MK2and mediates the phosphorylation and functions of MK2in vitro and in vivo. In the present study, we in the first explore the function of P38aMAPK on meiotic spindle organization and chromosome assignment in mouse oocytes, and understand the mechanism of aneuplord in the mammal oocyte.
Part one:p38a MAPK Is a MTOC-Associated Protein Regulating Spindle Assembly, Spindle Length and Accurate Chromosome Segregation during Mouse Oocyte Meiotic Maturation
The assembly of a functional bipolar spindle is critical for accurate chromosome segregation in mammalian oocytes. The size, shape and length of the spindle is relatively constant in one cell during spindle, which is regulated by motor proteins, protein kinase, microtubel-associated protein and pole-associated protein. The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK) and p38, all of which are highly conserved in all eukaryotic systems. p38mitogen-activated protein kinase (p38MAPK) is one of the three major members of the MAPK family that regulates cellular responses including apoptosis, cell proliferation and immune response as well as cell growth, differentiation, and cytoskeletal rearrangements. In the present study, we used the mouse oocytes as the model, investigating the roles of p38MAPK in the spindle assembly and spindle checkpoint activation, which enriches our knowledge about this cellular event in meiosis and provides the theoretical basis for the mechanisms forming the aneuploidy in mammalian oocytes.
Methods Immature oocytes were collected from ovaries of6-week-old female ICR mice. Firstly, we examined the expression and subcellular localization of this protein by immunoblotting analysis and immunofluorescence; next, we investigate the space localzation bwtween p38MAPK and MTOC in oocytes treated with Taxol。 Furthermore, we examined the phnotype in oocytes by special P38a inhibitor treatment and P38mophornilo injection. In addtion, we tested a possible interaction between endogenous p-p38a and p-MK2by co-immunoprecipitation with p-p38a antibody in mouse MI oocytes extracts and then performed immunoblot analysis with the anti-p-MK2antibody. Chromosome spreading was done for checking aneuploid in MII oocytes. At last, BubRl signal was detected in p38aMO-injected oocytes.
Results
1. Expression and subcellular localization of phospho-p38aMAPK (p-p38a) during mouse oocyte meiotic maturation The expression level of p-p38a remained stable from GV to MI stage, and then become reduced at the ATI and MII stages by Westernbloting. At the GV stage, p-p38a was localized in the germinal vesicle; after GVBD, multiple bright foci of p-p38a were detected near the individual chromosomes. By Pro-MI when chromosomes began to migrate to the equator of the spindle, p-p38a was gradually translocated to the spindle poles. Notably, p-p38a were specifically concentrated at the spindle poles at the MI stage. No evident signals of p-p38a were labeled in anaphase/telophase, however, p-p38a reappeared at the spindle poles in oocytes at the MII stage.
2.p-p38a colocalizes with y-tubulin at cytoplasmic MTOCs and spindle poles in mouse oocytes. p-p38a was colocalized with y-tubulin at the spindle poles and cytoplasmic MTOCs in oocytes at MI and MII stages. Taxol was employed for oocyte treatment. In this case, p-p38a was specifically colocalized with y-tubulin at the poles of the abnormal spindles, the center of MTOCs, and at the center of the cytoplasmic asters in oocytes at Pro-MI, MI and MII stages. Specifically, p-p38a was strictly colocalized with a broad "C"-shaped y-tubulin configuration unattached at the poles.
3.Depletion of p38α resulted in abnormal spindles and misaligned chromosomes To explore the roles of p38α, we employed a morpholino-based gene-silencing approach to deplete p38a in oocytes. Using a specific p38a-targeting morpholino (termed p38a-MO), we successful depleted most of the endogenous p-p38a protein in oocytes. P-p38a depletion did not affect meiotic cell cycle progression and rates of polar body extrusion. Down-regulation of p-p38a resulted in significant defects in spindle formation and chromosomes alignment. Aberrant spindle organization included elongated spindles and various defects in poles including multipolar spindles and disintegrated spindle poles. The rate of abnormal spindle formation in the p38aMO-injected group was74.4%(128/172), which was considerably higher than that of the control MO-injected group21.3%(40/188), x2=101.919, P<0.001. P38α-depleted oocytes displayed severe defects in chromosome alignment, showing lagging chromosomes and irregularly scattered chromosomes. The incidence of misaligned chromosomes in the p38aMO-injected group was up to75%(129/172), much higher than that in the control group (10.10%(19/188),(x2=156.241, P<0.001).
4. P38a depletion may compromise meiotic spindle organization and chromosome alignment via the p38α/MK2signaling pathway. Firstly, we tested a possible interaction between endogenous p-p38a and p-MK2by coimmunoprecipitation. p-MK2was clearly decreased in the p38a-depleted oocytes compared with the control group, indicating that p38a could phosphorylate and activate MK2. Subsequently, we examined the p-MK2localization after p38a depletion and demonstrated that p-MK2was dissociated from the spindle poles and chromosomes, and were scattered in the cytoplasm in the p38a-depleted oocytes.
5. p38a is required for recruitment of y-tubulin to MTOCs and stabilization of spindle bipolarity. In p38a-depleted MI oocytes, spindle organization was disrupted in over70%of oocytes (n=114). The typical defects in spindle assembly included aberrant poles (58.2%, n=114), with striking additional small poles around spindles or near the main poles. Significant γ-tubulin signals were observed in the cytoplasm in p38a-depleted MI oocytes. Moreover, MTOC asters labeled with y-tubulin loosely dispersed within the abnormal spindles or in the cytoplasm in MI oocytes, which indicates that activation of p38a is required for recruitment of cytoplasmic MTOCs to spindle poles. The oocytes with abnormal spindles displayed multiple bright foci loosely dispersed at the defective spindle poles. When treated with cold, microtubule-nucleating components became dissociated from the spindle ends in the p38a-depleted oocytes. Additionally, the minus ends of kinetochore microtubules were not tethered at the spindle poles. p38a was colocalized with Plkl at MTOCs and spindle poles in oocytes treated with taxol and in control oocytes. In p38a-depleted oocytes, Plkl was either completely dissociated from the spindle poles and chromosomes or Plkl signals became faint.
6. P38a and Eg5play opposite roles in determining spindle tension and length P38a-depleted oocytes displayed elongated spindles and increased tension between homologous kinetochores in pole-defocused spindles. The distance between homologous kinetochores in pole-defocused MI spindles of p38a-depleted oocytes (2.88μm±0.05, n=10spindles,64kinetochore pairs) was significantly increased compared to that of control oocytes (0.99μm,±0.04, n=8spindles,52kinetochore pairs) F=21.567, P<0.001, indicating that the tension between homologous kinetochores in pole-defocused spindles was notably increased. To quantify spindle length, we measured the spindle length as shown in the schematic. The spindles were50%longer in p38aMO-injected oocytes than those of control MO-injected oocytes (23.3±3.02μm). Interesting, spindle elongation was observed in Eg5-overexpressed oocytes (37.80±2.09μm). In contrast, monopolar spindles were observed in Eg5knocked-down oocytes. We further injected oocytes with both p38αMO and Eg5MO. As expected, in p38αMO and Eg5MO co-injected oocytes (25.3±2.72μm), bipolar spindle morphology and length were rescued.
7. P38a depletion affected the distribution of Eg5along microtubules in abnormal spindles. The results indicated that Eg5or dynein depletion did not affect the localization of p38a, and p38a was still localized at spindle poles. However, p38a depletion significantly affected the distribution of Eg5along the spindles. Eg5was localized at microtubules and concentrated at spindle poles in control MO injected oocytes. In Eg5-overexpressed oocytes, Eg5was localized to microtubules but not concentrated at the poles. However, in p38α-depleted oocytes, the expression of Eg5was decreased at poles, but increased on the spindle microtubules, notably at the midzone of the spindle. In some spindles, Eg5signal was only detected in half of the whole spindle.
8. P38a depletion inactivated SAC and increased frequencies of aneuploidy. We observed that the ratio of polar body extrusion (PBE) in p38a-depleted oocytes (78.2%, n=234) was not different from that of the control group (79.8%, n=196). However, p38a-delepted MI and MII oocytes showed obviously misaligned chromosomes. Thus, chromosome spreads were performed in p38a-depleted MII oocytes. Our results showed that p38a-depleted MII oocytes typically displayed incorrect numbers (more or less than20) of univalents. The frequency of aneuploidy of p38a-depleted MII oocytes42.4%(14/33) was significantly higher than that in control oocytes (4.8%(1/21))x2=9.07, P=0.003, P<0.05. We observed that BubRl was enriched at numerous kinetochores in control MO injected oocytes at the prometaphase stage. However, no BubRl signal was detected in p38aMO-injected oocytes.
Summary
1. P38a is an important component of MTOC, which regulates spindle assembly. P38a specifically colocalized with y-tubulin and Plkl at the center of MTOCs and spindle poles. Knockdown of p38a lead to the disperse of y-tubulin and Plkl at spindle pole, which support that p38a regulate spindle and microtuble nuclear and recruit y-tubulin to MTOC. Depletion of p38a by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes probably via MK2dephosphorylation.
2. P38a stablized the spindle and spindle pole in mouse oocyte. Depletion of p38a led to significant spindle pole defects, spindle elongation, non-tethered kinetochore microtubules and increased microtubule tension. The disruption of spindle stability was coupled with decreased y-tubulin and Plkl at MTOCs, which indicate that P38a connected the pole microtuble and MTOC with the negative of microtuble. The unclustered pole microtuble and kinetochore microtuble can not be assembled into normal spindle pole after P38a, which indicate that P38a is an important component of pole-associated proteins.
3. p38a antagonize the Eg5by indirectly producing a counteracting force, which regulate the tension and length of the spindle. Depleted-P38a led to inccreased spindle length and microtubel tension. Overexpression of Eg5, a conserved motor protein, also caused spindle elongation, and its morpholino injection almost completely rescued spindle elongation caused by p38a depletion, the presence of an antagonistic relationship between Eg5and p38a in mouse oocytes. Given that Eg5generates an outward force by sliding antiparallel microtubules, we considered the possibility that p38a may affect other minus proteins, which generate an inward force and partially antagonized Eg5. Thus, Eg5could be responsible for spindle elongation with increased tension in the absence of p38a. P38a depletion led to imbalance in force between spindle poles, which may be explained the abnormal poles in the absence of p38a.
4. p38a play an important pole in the formation of anueploid oocytes by regulating the spindle assemble checkpoint protein on kinetochores in mouse oocytes. Impairment of p38a disturbed the localization of BubRl at the kinetochores of chromosomes and compromised the SAC, which caused misaligned chromosome and subsequent aneuploidy in mouse oocyte meiosis.
Part two MK2Regulates Spindle Assembly and Accurate Chromosome Segregation during porcine Oocyte Meiotic Maturation
Methods
Porcine COCs were collected for culture in vitro. Firstly, the cumulus cell expansion and meiotic maturation of COCs were checked after the specific MK2inhibitor CMPD1treatment. Next, the meiotic maturation of DOs were investigated after CMPD1treatment. MK2were accurately located in spindle and chromosome、of porcine oocytes by immunofluorescence. To further clarify the correlation among MK2, MTOC, Crest and Plkl, taxol was employed for oocyte treatment. Furthermore, the spindle formation and chromosome alignment were explored after depletion of MK2by CMPD1treatment and MK2antibody injection.
Results
1. The effect of CMPD1on the cumulus expansion of porcine COCs After porcine COCs were cultured for24h in TMP-199with FSH,85.7%(281/328) of COCs in control were examined as cumulus expansion. High density CMPD1(30μmol/L) significantly inhibited the cumulus expansion (3.3%(8/242));12.5%(30/240)&24.39%(80/328) were expanded in the presence of20μmol/L and10μmol/L(χ2=546.22, P<0.001), which indicate that CMPD1inhibits porcine cumulus expansion dependly on the concentration of CMPD1.
2. Evaluating the effect of CMPD1on porcine COCs meiotic maturation. CMPD1dose not affect the meiotic recover of porcine oocyte, however, CMPD1affects the extrusion of the first polar body(pbl). After porcine COCs were cultured for42h in TMP-199with FSH, the rate of pbl extrusion in porcine oocytes in30,20,10μmol/L showed as0%;6.25%(11/176)&37.9%(50/132), which significantly decreased in comparison with the control (63.16%(60/95),χ2=178.17, P<0.001). Thus, CMPD1inhibit the pbl extrusion in porcine oocytes dependly on the concentrtion of CMPD1.
3. Evaluating the effect of CMPD1on meiotic maturation of porcine denude oocytes. CMPD1dose not affect the the meiotic recover of porcine oocyte but the pb1extrusion. The rate of pb1extrusion of porcine oocytes in30,20,10μmol/L showed as0%(0/136);5.41%(10/185)&19.48%(30/154), which significantly decreased in comparison with the control54.46%(55/101),χ2=150.055, P<0.001. Moreover, the rate of pbl extrusion in DO treated with10μmol/L CMPD1siginificantly decreased compared with the control (54.46%(55/101)).
4. The subcellular location of p-MK2during porcine oocyte meiotic maturation. After germinal vesicle breakdown (GVBD), p-MK2was localized along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions. When oocytes progressed to MI, homologous chromosomes aligned at the equatorial plate, and p-MK2localized at the spindle plus ends and chromosomal axes. At anaphase I, the homologous chromosomes were segregated, and the p-MK2signals disappeared from chromosomes and localized to the equatorial region between the separating chromosomes. At MII, the p-MK2signals again translocated to the spindle plus ends and was located between Crest and sister chromatids. P-MK2was partly colocalized with Plkl on chromosome and microtubles or was located between Crest and sister chromatids. P-MK2was partly colocalized was y-tubulin on spindle.
5. MK2inhibitor CMPD1affect the spindle assembly and chromosome assignment in porcine oocytes. Down-regulation of MK2resulted in significant defects in spindle formation and chromosomes alignment. Aberrant spindle organization included collapsed spindle surrounded by dispersed chromsomes and irregularly scattered spindle. The rate of abnormal spindle formation in CMPD1treatment group was67.78%(61/90), which was considerably higher than that of the control group18.35%(20/109);χ2=49.90, P<0.001. P38a-depleted oocytes displayed severe defects in chromosome alignment, showing lagging chromosomes and irregularly scattered chromosomes. The incidence of misaligned chromosomes in CMPD1treatment group was up to63.33%(57/90), much higher than that in the control group21.10%(23/109),χ2=36.560, P<0.001.
6. MK2antibody injection disrupted spindle assemble and chromosome assignment in porcine oocytes. The rate of abnormal spindle formation in antibody-injected group was66.9%(79/118), which was considerably higher than that of the control group21.0%(42/200);(χ2=66.47, P<0.001); the incidence of misaligned chromosomes in antibody-injected group was up to70.34%(83/118), much higher than that in the control group_(23.5%(47/200χ2=67.37, P<0.001).
Summary
1. MK2play an important role in cumulus expansion of porcine oocytes induced by FSH through the MK2on the cumulus. MK2inhibitor CMPD1significantly inhibited the cumulus expansion of COCs.
2. MK2regulates spindle assemble and chromosome assignment in porcine oocytes. collapse spindles surrounded by the chromosomes is a specific phenotype, which indicates that MK2can connected microtuble with crest. The results from CMPD1treatment and antibody injection support the above views.
3. MK2regulates the procession of porcine oocytes during meiotic maturation. However, MK2dose not affect the spontaneous meiotic recovery in GV oocytes.
Conclusion
1. p38aMAPK regulate spindle, spindle pole organization and chromosome assignment. p-p38α was located at spindle pole and was colocalized with γ-tubulin at MTOC and cytoplasm aster; p38aMAPK can recuit Plk1and γ-tubulin to spindle pole and MTOC and promote the microtuble nucleation. p38aMAPK regulates kintochore microtuble polymerized and stablized the spindles and spindle poles. Thus, p38aMAPK is one component of MTOCs in mouse oocytes and also is a negative microtuble-associated protein.
2. p38a and Eg5are an antagonism force, which regulate spindle tension and length. p38a can regulate spindle tension and length through changing Eg5distribution at microtubule, but not affect the location of Dynein. p38a is an important kinase. It needs to be further investigated whether p38aMAPK regulates spindle tension through phosphrylating motor proteins.
3. p38a affects the spindle checkpoints BubR1in mouse oocytes and regulates chromosome assignment, which is an important mechanism about mouse aneuploid.
4. MK2promotes porcine comulus expandation through FSH induction on the cumulus. MK2also regulates spindle assemble, chromosome separation and connected the Crest with microtubles in porcine oocytes. MK2interupts the pbl extrusion, but not affect the spontanous meiotic recovery in porcine oocytes.
5. The location of p38a is charaterized with species distinction. p38a is localized at the cytoplasm of porcine oocytes, but at the spindle poles in mouse oocytes. However, the location of MK2in mouse is the same as in the porcine oocytes. In mouse oocytes, p38a is formed a compound with MK2and MK2is the downstream of p38aMAPK, which phosphrylates Mk2. p38a regulates spindles assemble and chromosome assignments through p38aMAPK-MK2pathway. However, the relatation between p38aMAPK and MK2in the porcine oocytes is different from the mouse oocytes. Thus the mechanism about MK2in porcine oocytes need to further be explored.
引文
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