小鼠卵母細(xì)胞減數(shù)分裂過(guò)程中LIMK1參與調(diào)控Aurora-A在紡錘體的定位

姜秀穎，王思敏，周 晴，翁 靜，馬 偉

(首都醫(yī)科大學(xué) 基礎(chǔ)醫(yī)學(xué)院 組織學(xué)與胚胎學(xué)教研室, 北京 100069)

胚胎的非整倍體性(aneuploidy)是臨床不育、自發(fā)性流產(chǎn)和先天性出生缺陷的最常見(jiàn)原因，這種染色體組成異常主要由母親卵母細(xì)胞減數(shù)分裂進(jìn)程中染色體分離錯(cuò)誤所導(dǎo)致[1- 2]。染色體的運(yùn)動(dòng)和分離由紡錘體牽引，紡錘體結(jié)構(gòu)的異常會(huì)影響染色體分離的精確性。在哺乳動(dòng)物卵子發(fā)生早期，中心粒退化，缺乏中心體結(jié)構(gòu)，減數(shù)分裂紡錘體的形成依賴于無(wú)中心粒的微管組織中心(microtubule organizing centers, MTOC)[3- 4]。MTOC結(jié)構(gòu)由核心蛋白和調(diào)節(jié)蛋白組成，前者包括結(jié)構(gòu)成分pericentrin和微管成核因子γ-tubulin；調(diào)節(jié)蛋白則主要指一些蛋白激酶，如Aurora-A激酶等，促進(jìn)MTOC的結(jié)構(gòu)和功能完全成熟[5- 7]。

LIM kinase 1 (LIMK1)是一種絲氨酸(Ser)/蘇氨酸(Thr)蛋白激酶，其活化依賴于Thr508殘基的磷酸化，活化的LIMK1通過(guò)調(diào)控F-actin的聚合而參與細(xì)胞運(yùn)動(dòng)、胞質(zhì)分裂和囊泡運(yùn)輸?shù)壬磉^(guò)程[8]；同時(shí)也參與維持微管的穩(wěn)定性，促進(jìn)星狀微管的形成和紡錘體的準(zhǔn)確定位[9]。關(guān)于LIMK1在卵母細(xì)胞減數(shù)分裂過(guò)程中表達(dá)模式及其與MTOC蛋白之間的關(guān)系仍無(wú)研究報(bào)道。

1 材料與方法

1.1 材料

清潔級(jí)21日齡CB6F1雌性小鼠(雄性C57BL6與雌性BALB/C的雜交一代)[北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司，許可證編號(hào)SCXK(京)2012- 0001]；兔抗pLIMK1Thr508抗體(Millipore公司)；小鼠抗acetylated-tubulin抗體、小鼠抗Aurora-A抗體和牛血清白蛋白(bovine serum albumin, BSA)(Sigma-Aldrich公司);小鼠抗pericentrin抗體(BD Biosci-ences公司)；LIMKI抑制劑BMS- 3(Synkinase公司)；孕馬血清促性腺激素(pregnant mare serum gonadotropin,PMSG)(寧波第二激素廠)；胎牛血清(fetal bovine serum, FBS)(Gibco公司)；防熒光淬滅封片劑(mounting medium with DAPI)(Vector Lab公司)。

1.2 方法

1.2.1 卵母細(xì)胞的收集和培養(yǎng)：給CB6F1代雌性小鼠腹腔注射10 IU PMSG，44～48 h后使用CO2窒息法對(duì)小鼠實(shí)行安樂(lè)死，隨后收集卵丘-卵母細(xì)胞復(fù)合體(cumulus cell-oocyte complexes, COC)進(jìn)行培養(yǎng)?；A(chǔ)培養(yǎng)液為Minimum Essential Medium(MEM)，培養(yǎng)過(guò)程中添加10% FBS和3 mg/mL BSA。體外培養(yǎng)0、2、8、10和16 h，分別對(duì)應(yīng)減數(shù)分裂前期(即生發(fā)泡期，germinal vesicle，GV)、生發(fā)泡破裂期(germinal vesicle breakdown, GVBD)、第1次減數(shù)分裂中期(metaphase Ⅰ, MⅠ)、第1次減數(shù)分裂后期(anaphase Ⅰ, AⅠ)和末期(telphase Ⅰ, Tel Ⅰ)以及第2次減數(shù)分裂中期(metaphase Ⅱ, MⅡ)。

1.2.2 免疫熒光染色(immunofluorecent staining)：室溫條件下，將脫去卵丘的卵母細(xì)胞在1%多聚甲醛(paraformaldehyde,PFA)/0.5% Triton X- 100中固定45 min，漂洗后在10%山羊血清中封閉1 h，隨后在一抗溶液中孵育，4 ℃、過(guò)夜。細(xì)胞漂洗后繼續(xù)在Alexa Flour- 488或Alexa Flour- 594標(biāo)記的相應(yīng)二抗溶液中避光孵育45 min，充分漂洗后移至干凈的載玻片上并以含有DAPI的防淬滅封片劑封片。樣品用Olympus熒光顯微鏡進(jìn)行觀察并分析減數(shù)分裂中pLIMK1Thr508和Aurora-A的時(shí)空分布相關(guān)性。試驗(yàn)中抗體主要包括：兔抗pLIMK1Thr508(1∶1 000)，小鼠抗acetylated-tubulin(1∶10 000)和小鼠抗Aurora-A(1∶3 000)。

1.2.3 BMS- 3處理：1 mmol/L BMS- 3儲(chǔ)存在二甲基亞砜(DMSO, Sigma-Aldrich公司)中，并在培養(yǎng)基(MEM/BSA/10% FBS)中稀釋至實(shí)驗(yàn)濃度0.001 mmol/L。正常培養(yǎng)至MⅠ期的卵母細(xì)胞在0.001 mmol/L BMS- 3中處理1 h后固定染色，觀察pLIMK1Thr508和Aurora-A的空間分布以及紡錘體結(jié)構(gòu)的變化。

1.3 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 pLIMK1Thr508在小鼠卵母細(xì)胞在減數(shù)分裂進(jìn)程中的亞細(xì)胞定位及其與Aurora-A的相關(guān)性分析

免疫熒光染色分析表明在減數(shù)分裂前期，卵母細(xì)胞內(nèi)Aurora-A沒(méi)有任何特殊聚集(圖1B)，此時(shí)pLIMK1Thr508的熒光信號(hào)亦比較弱，主要分布在生發(fā)泡中(圖1C: 箭)。在臨近減數(shù)分裂重啟時(shí)，pLIMK1Thr508離開(kāi)生發(fā)泡并以一致密點(diǎn)狀聚集在胞質(zhì)中，Aurora-A信號(hào)出現(xiàn)并與pLIMK1Thr508高度重合(圖1F, G: 箭)。在生發(fā)泡完全破裂后，染色質(zhì)凝集成為單個(gè)的棒狀染色體(圖1I)，pLIMK1Thr508和Aurora-A由單一點(diǎn)狀裂解為多個(gè)片狀信號(hào)并圍繞在染色體周圍，在此過(guò)程中兩種信號(hào)保持重合(圖1J, K, L：箭)。在第1次減數(shù)分裂中期(metaphase I, MI)，所有的染色體整齊排列在赤道板中央(圖1M)，pLIMK1Thr508和Aurora-A同時(shí)高度聚集，呈“C”形或“O”形特異地定位于染色體陣列的兩側(cè)，即推測(cè)中紡錘體的兩極部位(圖1N, O, P：箭)，兩者在胞質(zhì)中也有散在點(diǎn)狀分布(圖1N, O, P：箭頭)。在第1次減數(shù)分裂后期(anaphase I, AI)到末期(telphase I, TelI)過(guò)程中，隨著同源染色體相互分離(圖1Q)，pLIMK1Thr508離開(kāi)紡錘體區(qū)域，聚集在收縮環(huán)部位(圖1S：箭)，提示pLIMK1Thr508可能與胞質(zhì)分裂的完成相關(guān)；Aurora-A信號(hào)變?nèi)?，彌散地分布在微管所在部?圖1R：箭)。在卵母細(xì)胞成熟至第2次減數(shù)分裂中期(metaphase II, MII)時(shí)，第一極體(first polar body, 1st PBD)被排出(圖1U：箭)，在其附近的皮質(zhì)區(qū)染色體重新排列整齊(圖1U)，此時(shí)pLIMK1Thr508和Aurora-A又同時(shí)聚集在染色體陣列兩側(cè)，即紡錘體兩極部位(圖1V,W,X：箭)，在胞質(zhì)中也有致密的點(diǎn)狀分布(圖1V,W,X：箭頭)。pLIMK1Thr508和Aurora-A在紡錘體兩極部位的共定位模式提示LIMK1可能通過(guò)調(diào)控Aurora-A的定位和表達(dá)而參與紡錘體組裝過(guò)程，而pLIMK1Thr508在收縮環(huán)部位的聚集也提示LIMK1活性可能還與胞質(zhì)分裂密切相關(guān)。

2.2 抑制LIMK1活性影響Aurora-A的空間分布和紡錘體形成

正常培養(yǎng)至MI期的卵母細(xì)胞在用1 μmol/L BMS- 3處理1 h后，pLIMK1Thr508和Aurora-A的空間分布發(fā)生了明顯改變，失去了對(duì)稱的雙極定位格局，以類似于前中期的模式圍繞在染色體周圍(圖2A. f, g：箭)，此時(shí)，染色體非線性排列而是凌亂地聚集在一起(圖2A. e; B. e)，微管并沒(méi)有組織形成雙極對(duì)稱的紡錘體結(jié)構(gòu)，而是成球狀圍繞在染色體組周圍(圖2B. f：方框)。統(tǒng)計(jì)分析證實(shí)，在BMS- 3處理組內(nèi)表現(xiàn)出pLIMK1Thr508和Aurora-A非雙極定位的細(xì)胞數(shù)目顯著高于對(duì)照組(P<0.01)(圖2C, D)，而具有非雙極對(duì)稱紡錘體的細(xì)胞數(shù)目亦在BMS- 3組顯著高于對(duì)照組(P<0.01)(圖2E)。上述數(shù)據(jù)說(shuō)明LIMK1活性通過(guò)調(diào)控Aurora-A的空間分布而影響MTOC結(jié)構(gòu)形成和紡錘體組裝。

3 討論

Aurora-A激酶參與調(diào)節(jié)體細(xì)胞有絲分裂進(jìn)程中中心體的復(fù)制和分離、染色體的中板聚合以及雙極紡錘體的形成等多個(gè)事件[5]，并在卵母細(xì)胞內(nèi)參與MTOC功能和雙極紡錘體形成的調(diào)控，是一種MTOC調(diào)節(jié)蛋白，其活性異常或表達(dá)缺失會(huì)影響MTOC形成和紡錘體組裝過(guò)程[6- 7]。在有絲分裂過(guò)程中LIMK1活性能夠促進(jìn)Aurora-A在Ser288位點(diǎn)磷酸化，使其全面活化[10]，說(shuō)明LIMK1與Aurora-A的功能相關(guān)。本項(xiàng)研究進(jìn)一步發(fā)現(xiàn)pLIMK1Thr508與Aurora-A在小鼠卵母細(xì)胞減數(shù)分裂過(guò)程中共同定位于紡錘體兩極，抑制LIMK1活性會(huì)導(dǎo)致Aurora-A定位和紡錘體結(jié)構(gòu)異常，提示LIMK1活性可能通過(guò)調(diào)控Aurora-A的活化和極性定位而參與紡錘體的組裝和維持。在卵母細(xì)胞減數(shù)分裂末期，胞質(zhì)分裂的完成依賴于收縮環(huán)的有效活動(dòng)，而收縮環(huán)結(jié)構(gòu)的形成依賴于絲狀肌動(dòng)蛋白(filament actin, F-actin)聚合和解聚的動(dòng)態(tài)平衡[11- 12]，以往研究證實(shí)LIMK1能夠磷酸化微絲切割蛋白cofilin，抑制其解聚F-actin的活性，誘導(dǎo)F-actin聚合[13- 14]。本項(xiàng)研究發(fā)現(xiàn)pLIMK1Thr508高度聚集在收縮環(huán)部位，提示卵母細(xì)胞內(nèi)LIMK1-cofilin通路可能通過(guò)調(diào)節(jié)收縮環(huán)的功能而促進(jìn)胞質(zhì)分裂過(guò)程的有序完成。總之，本項(xiàng)研究初步證實(shí)卵母細(xì)胞內(nèi)pLIMK1Thr508是一種MTOC相關(guān)蛋白，LIMK1活性可能通過(guò)參與MTOC形成和胞質(zhì)分裂等環(huán)節(jié)而調(diào)控減數(shù)分裂進(jìn)程，研究結(jié)果為進(jìn)一步揭示卵母細(xì)胞內(nèi)染色體分離的調(diào)控機(jī)制和認(rèn)識(shí)非整倍體胚胎的形成提供了依據(jù)。

DNA was labeled in blue,whereas pLIMK1Thr508in red, Aurora-A in green; pLIMK1Thr508concentrated in the germinal vesicle (C, arrow)with no signal of Aurora-A(B) at GV stage; upon GVBD, pLIMK1Thr508aggregated as a single dense dot in the vicinity of nuclear, and co-localized with Aurora-A(G,F,arrow); at G,V,B,D, pLIMK1Thr508and Aurora-A (J,K,arrow)concentrated as multi foci around the chromosomes; at MI and MII, pLIMK1Thr508(O,W, arrow) was co-localized with Aurora-A(N,V, arrow) on spindle poles, pLIMK1Thr508(O,W, arrowhead) which scattered as dots in cytoplasm was co-localized with Aurora-A(N,V, arrowhead); during Tel I, pLIMK1Thr508(S, arrow) concentrated on the cleavage furrow, while Aurora-A (R) loosely congressed on spindle

圖1pLIMK1Thr508和Aurora-A在卵母細(xì)胞減數(shù)分裂中的亞細(xì)胞定位模式
Fig1Sub-cellularlocalizationofpLIMK1Thr508andAurora-Ainoocytemeiosis(×400)

DNA was labeled in blue,whereas pLIMK1Thr508in red, Aurora-A and acetylated tubulin in green; A.abnormal pLIMK1Thr508(g) and abnormal Aurora-A (f) in oocytes cultured with BMS-3 for 8 hours(×400); B.in BMS-3-treated oocytes, microtubules were not organised into a bipolar structure (f) and pLIMK1Thr508(g) were not recruited to spindle poles, the number of oocytes with (×400); C.non-polar location of pLIMK1Thr508; D.non-polar location of Aurora-A; E.disrupted spindle; was significantly higher in BMS-3 treatment group;*P<0.01 compared with DMSO group

圖2小鼠卵母細(xì)胞中抑制LIMK1活性破壞Aurora-A的空間分布和紡錘體形成

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