張楠，張玨，林戈,

綜 述

哺乳動物卵母細(xì)胞的DNA損傷與修復(fù)研究進(jìn)展

張楠1，張玨2，林戈1,2

1. 中南大學(xué)基礎(chǔ)醫(yī)學(xué)院生殖與干細(xì)胞工程研究所，長沙 410000 2. 中信湘雅生殖與遺傳?？漆t(yī)院，長沙 410000

DNA損傷是影響配子發(fā)生和胚胎發(fā)育的關(guān)鍵因素之一。卵母細(xì)胞容易被各種內(nèi)外源因素(如活性氧、輻射、化療藥物等)誘發(fā)DNA損傷。目前研究發(fā)現(xiàn)，對于各類DNA損傷，各發(fā)育階段的卵母細(xì)胞能夠做出相應(yīng)的DNA損傷反應(yīng)，通過復(fù)雜的機制對DNA進(jìn)行修復(fù)或者啟動細(xì)胞凋亡。相比于進(jìn)入生長階段的卵母細(xì)胞，原始卵泡卵母細(xì)胞更容易被DNA損傷誘導(dǎo)凋亡。DNA損傷不易誘導(dǎo)卵母細(xì)胞減數(shù)分裂成熟進(jìn)程停滯，然而攜帶DNA損傷的卵母細(xì)胞的發(fā)育能力明顯下降。在臨床上，衰老、放療和化療是導(dǎo)致女性卵母細(xì)胞DNA損傷、卵巢儲備降低和不孕的常見原因。為此，人們嘗試了能夠減輕卵母細(xì)胞DNA損傷和增強DNA修復(fù)能力的多種方法，試圖保護卵母細(xì)胞。本文對哺乳動物的各發(fā)育階段卵母細(xì)胞的DNA損傷與修復(fù)的相關(guān)研究進(jìn)行了梳理和總結(jié)，并討論了其潛在的臨床價值，以期為生育力保護提供新的策略。

卵母細(xì)胞；DNA損傷；DNA修復(fù)；DNA損傷反應(yīng)

DNA作為遺傳信息的核心，是細(xì)胞存活和發(fā)揮功能的基石。對于生殖細(xì)胞而言，DNA的完好性是其正常發(fā)育和形成健康胚胎的基礎(chǔ)[1]。然而，DNA是脆弱的，許多有害物質(zhì)都會造成細(xì)胞DNA損傷。細(xì)胞能夠通過復(fù)雜的DNA修復(fù)機制對損傷的DNA進(jìn)行修復(fù)。如果損傷的DNA沒有被成功修復(fù)，則有可能導(dǎo)致基因突變、細(xì)胞功能改變和疾病的發(fā)生[2]。哺乳動物卵母細(xì)胞的質(zhì)量是早期胚胎發(fā)育能力的關(guān)鍵決定因素[3]。卵母細(xì)胞的DNA損傷可能導(dǎo)致機體的生殖功能下降甚至喪失[1,4]。近年來，關(guān)于卵母細(xì)胞的DNA損傷與修復(fù)機制的研究逐漸成為一個熱點。在各種DNA損傷類型中，對DNA雙鏈斷裂(double-strand breaks，DSBs)的研究是最多的。目前研究認(rèn)為，對于各種內(nèi)外源因素誘導(dǎo)的DNA損傷，哺乳動物卵母細(xì)胞是敏感的，能夠及時啟動DNA損傷反應(yīng)(DNA damage response，DDR)，發(fā)生DNA修復(fù)或凋亡等一系列活動[5～7]。這對于保障卵母細(xì)胞質(zhì)量及早期胚胎的正常發(fā)育是至關(guān)重要的。本文對哺乳動物的各發(fā)育階段卵母細(xì)胞的DNA損傷與修復(fù)機制的研究現(xiàn)狀進(jìn)行了闡述，強調(diào)了卵母細(xì)胞DNA損傷在衰老和放化療導(dǎo)致的生育力下降中的重要作用，并簡要總結(jié)了目前關(guān)于減輕卵母細(xì)胞DNA損傷和改善卵母細(xì)胞質(zhì)量的可能方法。

1 體細(xì)胞的經(jīng)典DDR與DNA修復(fù)機制

細(xì)胞的DNA損傷可由各種外源性或內(nèi)源性因素引起，前者即環(huán)境因素，如紫外線、電離輻射、毒性化學(xué)物質(zhì)，后者即細(xì)胞內(nèi)代謝活動的產(chǎn)物，如氧化呼吸、脂質(zhì)過氧化等活動產(chǎn)生的活性氧(reactive oxygen species，ROS)。此外，DNA損傷也可以自發(fā)產(chǎn)生[2]。DNA損傷的形式包括堿基改變、單鏈斷裂、DSBs、加合物病變、鏈間交聯(lián)(interstrand crosslinks，ICLs)等[8]。這些損傷可能干擾DNA復(fù)制或轉(zhuǎn)錄過程進(jìn)而損害細(xì)胞功能。細(xì)胞通常能夠積極地做出DDR以應(yīng)對DNA損傷。DDR途徑由多種蛋白質(zhì)組成。根據(jù)功能的不同，這些蛋白質(zhì)可分為傳感器、調(diào)解器、轉(zhuǎn)導(dǎo)器和效應(yīng)器。DDR涉及許多細(xì)胞反應(yīng)，包括細(xì)胞周期停滯、染色質(zhì)重塑、損傷修復(fù)和細(xì)胞凋亡，是應(yīng)對刺激較全面的細(xì)胞反應(yīng)之一[2,8]。細(xì)胞主要在G1/S期和G2/M期激活DDR機制。共濟失調(diào)毛細(xì)血管擴張突變蛋白(ataxia telangiectasia mutated，ATM)和ATM與Rad3相關(guān)蛋白(ATM and Rad3 related，ATR)是重要的DNA損傷檢查點激酶。在G1期，ATM和ATR激酶被招募至DNA損傷位點并發(fā)生磷酸化，隨即激活下游的細(xì)胞周期檢查點激酶1(checkpoint kinase 1，CHK1)和細(xì)胞周期檢查點激酶2(checkpoint kinase 2，CHK2)[9～11]。CHK1和CHK2激酶繼續(xù)激活下游效應(yīng)器p53。在p53的介導(dǎo)下，p21結(jié)合并抑制了細(xì)胞周期依賴性激酶(cyclin- dependent kinase，CDK)的活性，從而使細(xì)胞周期暫停[12,13]。在G2期，DNA損傷位點同樣會招募并激活A(yù)TM/ATR激酶和CHK1/CHK2激酶。不同的是，CHK1/CHK2激酶通過抑制細(xì)胞分裂周期因子25(cell division cyclin 25，CDC25)磷酸酶進(jìn)而抑制CDK1的激活，從而使細(xì)胞周期停滯[13～15]。在細(xì)胞周期停滯期間，細(xì)胞將通過復(fù)雜的機制對損傷的DNA進(jìn)行修復(fù)。在DNA修復(fù)完成后，DNA損傷檢查點激酶發(fā)生去磷酸化，細(xì)胞周期恢復(fù)[13～15]。當(dāng)DNA損傷不能被完全修復(fù)時，p53將激活促凋亡基因如p53上調(diào)凋亡調(diào)節(jié)基因(p53-upregulated modu-lator of apoptosis，)和佛波醇-12-豆蔻酸-13-乙酸誘導(dǎo)蛋白1基因(phorbol-12-myristate-13-acetate- induced protein 1，/)的轉(zhuǎn)錄，從而誘導(dǎo)細(xì)胞凋亡[2,16,17]。

對于不同類型的DNA損傷，細(xì)胞能夠通過相應(yīng)的DNA修復(fù)機制進(jìn)行修復(fù)。DNA復(fù)制過程中發(fā)生的堿基錯配能夠通過錯配修復(fù)(mismatch repair，MMR)機制得到糾正；發(fā)生微小化學(xué)變化的堿基能夠通過堿基切除修復(fù)(base excise repair，BER)機制被去除；較大的DNA病變則可通過核苷酸切除修復(fù)(nucleotide excision repair，NER)機制被去除；DNA單鏈斷裂的修復(fù)過程涉及一組酶級聯(lián)反應(yīng)；同源重組(homologous recombination，HR)與非同源末端連接(non-homologous end joining，NHEJ)是修復(fù)DSBs的兩種機制；ICLs病變的去除機制涉及一組與范可尼貧血蛋白相關(guān)的復(fù)雜反應(yīng)[2,18]。普遍認(rèn)為，DSBs是DNA損傷類型中最嚴(yán)重的，可導(dǎo)致基因組重排和結(jié)構(gòu)變化，如缺失、易位、融合等[2,8,19]。DSBs的修復(fù)機制包括HR和NHEJ。在HR修復(fù)過程中，首先MRN復(fù)合物被招募至DSBs末端并對DNA末端進(jìn)行處理和切割以產(chǎn)生單鏈DNA[20,21]。之后，復(fù)制蛋白A(replication protein A，RPA)將單鏈DNA包裹，使其免受核酸酶作用并去除其二級結(jié)構(gòu)。在乳腺癌蛋白2(breast cancer 2，BRCA2)的介導(dǎo)下，RPA被DNA修復(fù)蛋白RAD51(DNA repair protein RAD51)替換。隨后，RAD51介導(dǎo)單鏈DNA侵入未受損的姐妹染色體[22,23]。最后在聚合酶、核酸酶、螺旋酶和其他分子的作用下，DNA進(jìn)行延伸并完成修復(fù)[24,25]。不同于HR，NHEJ是通過DNA連接酶將DSBs末端直接進(jìn)行連接。首先Ku70/Ku80蛋白識別并結(jié)合至DSBs末端，隨后招募并激活DNA依賴性蛋白激酶催化亞基蛋白(DNA-dependent protein kinase catalytic subunit，DNA-PKcs)[26,27]。之后，DNA-PKcs招募重組酶Artemis對DNA末端進(jìn)行處理，同時招募由X射線修復(fù)交叉互補蛋白4(X-ray repair cross-complementing protein 4，XRCC4)和DNA連接酶4(DNA ligase 4，LIG4)組成的蛋白復(fù)合物對DNA末端進(jìn)行連接[27,28]。HR發(fā)生在細(xì)胞周期的S期和G2期，利用未受損的姐妹染色體進(jìn)行修復(fù)，因而更加精準(zhǔn)。NHEJ是將DSBs的兩端直接連接在一起，雖不精準(zhǔn)，但可以在整個細(xì)胞周期運作[25,29]。普遍認(rèn)為，DNA損傷的累積和錯誤的DNA修復(fù)容易引起基因突變和染色體畸變，導(dǎo)致細(xì)胞功能的減退和喪失，進(jìn)而可能促進(jìn)衰老和疾病的發(fā)生[2,8,30]。因此，細(xì)胞保持其基因組的穩(wěn)定性和完整性是至關(guān)重要的。

2 各發(fā)育階段卵母細(xì)胞的DNA損傷與修復(fù)機制

2.1 原始卵泡卵母細(xì)胞

胎兒期的卵母細(xì)胞開始了第一次減數(shù)分裂，在前期的細(xì)線期至粗線期階段，同源染色體發(fā)生了聯(lián)會和重組事件。在同源染色體重組完成后，卵母細(xì)胞即停滯在第一次減數(shù)分裂前期的雙線期。在同源染色體聯(lián)會過程中，染色體內(nèi)主動發(fā)生DNA DSBs，從而允許同源非姐妹染色體之間進(jìn)行基因重組和形成染色體交叉。染色體交叉對同源染色體的中期對齊和后期正確分離至關(guān)重要[31,32]。卵母細(xì)胞的減數(shù)分裂重組過程受到了時間和空間的嚴(yán)格調(diào)控，重組過程中的DSBs修復(fù)失敗會導(dǎo)致卵母細(xì)胞凋亡或卵母細(xì)胞染色體錯誤[31～33]。哺乳動物出生后的卵母細(xì)胞與周圍的單層扁平顆粒細(xì)胞共同形成原始卵泡結(jié)構(gòu)。原始卵泡卵母細(xì)胞處于第一次減數(shù)分裂前期的雙線期，此時卵母細(xì)胞中的同源染色體通過染色體交叉連接在一起。在被招募進(jìn)入生長發(fā)育軌道前，原始卵泡卵母細(xì)胞長期維持在第一次減數(shù)分裂前期的雙線期，并保持代謝不活躍的狀態(tài)[34]。這一維持時間在小鼠()中可達(dá)數(shù)月，在人類()中則長達(dá)50年[35]。由于長期停滯在第一次減數(shù)分裂前期的雙線期，原始卵泡卵母細(xì)胞發(fā)生并累積DNA損傷的風(fēng)險較高。原始卵泡卵母細(xì)胞的DNA完好是卵母細(xì)胞后續(xù)發(fā)育的基本保障，此外原始卵泡庫代表著哺乳動物生命中全部的生殖儲備。因此原始卵泡卵母細(xì)胞對DNA損傷有著敏感和嚴(yán)格的監(jiān)控反應(yīng)機制，以能夠修復(fù)DNA損傷或清除存在損傷的卵母細(xì)胞，從而確保個體排出最優(yōu)質(zhì)的卵母細(xì)胞用于受精和胚胎發(fā)育。

大量動物實驗和臨床數(shù)據(jù)表明，輻射和化療藥物等外源性因素很容易誘導(dǎo)原始卵泡卵母細(xì)胞出現(xiàn)廣泛DNA損傷和發(fā)生凋亡[36～40]。在臨床上，化療和卵巢局部放療導(dǎo)致的原始卵泡庫的損耗容易誘發(fā)早發(fā)性卵巢功能不全(premature ovarian insufficiency，POI)、不孕或過早絕經(jīng)等疾病[36,37,41]。在體細(xì)胞DNA損傷時，p53是介導(dǎo)細(xì)胞凋亡的關(guān)鍵因子。與體細(xì)胞不同的是，由基因(人類為基因)編碼的反式激活p63蛋白(trans-activating p63，TAp63)是介導(dǎo)DNA損傷的卵母細(xì)胞發(fā)生凋亡的關(guān)鍵因子，TAp63特異性表達(dá)于原始卵泡和初級卵泡的卵母細(xì)胞中。是迄今為止發(fā)現(xiàn)的唯一參與卵母細(xì)胞DDR的家族成員[42～47]。人類基因功能獲得性突變導(dǎo)致卵母細(xì)胞凋亡，最終導(dǎo)致POI的發(fā)生[48]。當(dāng)原始卵泡卵母細(xì)胞發(fā)生DNA損傷時，在ATM激酶和CHK2激酶的調(diào)控下，TAp63被磷酸化激活。TAp63由封閉的、無活性的二聚體構(gòu)象轉(zhuǎn)變?yōu)殚_放的、有活性的四聚體構(gòu)象，其與DNA的結(jié)合親和力顯著提高[42,49]。磷酸化的TAp63繼而激活和等基因的轉(zhuǎn)錄和翻譯。之后PUMA和NOXA與促凋亡的BCL2相關(guān)的X蛋白(BCL2-associated X protein，BAX)和BCL2拮抗劑/殺傷蛋白(BCL2- antagonist/killer，BAK)相互作用，最終誘導(dǎo)細(xì)胞凋亡[36～40]。通過進(jìn)一步構(gòu)建、和等基因敲除的小鼠，研究發(fā)現(xiàn)經(jīng)γ輻射或化療藥物處理的基因敲除小鼠能夠維持原始卵泡數(shù)量，并生育正常的子代[38,40,45,50～52]。綜上所述，自然情況下，大部分原始卵泡卵母細(xì)胞在發(fā)生廣泛的DNA損傷時，選擇啟動凋亡程序以消除自身；然而當(dāng)人為阻斷其凋亡時，這些基因受損的卵母細(xì)胞能夠存活，甚至可以完成生育的使命，表明原始卵泡卵母細(xì)胞有能力對DNA損傷進(jìn)行有效修復(fù)。

在對于原始卵泡卵母細(xì)胞的DNA修復(fù)的相關(guān)研究中，關(guān)于DSBs修復(fù)的研究是最多的。由于原始卵泡卵母細(xì)胞長期停滯在第一次減數(shù)分裂前期，細(xì)胞中存在的姐妹染色體可以提供準(zhǔn)確修復(fù)的模板，因此原始卵泡卵母細(xì)胞主要通過復(fù)雜而精確的HR機制對DSBs進(jìn)行修復(fù)，以保障卵母細(xì)胞的基因組完整性。RAD51是重要的HR修復(fù)因子，Kujjo等[53]首次在小鼠的原始卵泡卵母細(xì)胞內(nèi)發(fā)現(xiàn)了RAD51。后續(xù)研究進(jìn)一步證明，小鼠原始卵泡卵母細(xì)胞通過激活A(yù)TM，磷酸化組蛋白H2AX(組蛋白H2A變體)的139位絲氨酸，即形成γH2AX位點，將RAD51定位于DNA斷裂位點，從而通過HR機制對γ輻射和化療藥物誘導(dǎo)的DSBs進(jìn)行快速修復(fù)[50,52]。乳腺癌蛋白1(breast cancer 1，BRCA1)也是HR修復(fù)因子家族的重要成員[25]。BRCA1在小鼠和人類的原始卵泡卵母細(xì)胞的DSBs修復(fù)中發(fā)揮關(guān)鍵作用，基因突變?nèi)巳旱脑悸雅輧錅p少[54,55]。NHEJ作為另一種DSBs修復(fù)機制，在原始卵泡卵母細(xì)胞中可能不發(fā)揮主要作用[52,56]。除DSBs損傷外，范可尼貧血互補群E型基因(Fanconi anemia complemen-tation group E，)在小鼠原始卵泡卵母細(xì)胞的ICLs損傷修復(fù)中發(fā)揮關(guān)鍵作用，基因敲除小鼠的原始卵泡幾乎耗竭[57]。關(guān)于原始卵泡卵母細(xì)胞的其他類型DNA損傷與修復(fù)機制有待進(jìn)一步探索。

2.2 生長階段的卵母細(xì)胞

在卵泡刺激素的作用下，原始卵泡被激活進(jìn)入生長發(fā)育軌道，成為生長卵泡。生長卵泡進(jìn)一步可分為初級卵泡、次級卵泡和竇卵泡。生長階段的卵母細(xì)胞依然維持在第一次減數(shù)分裂前期的雙線期。此階段的卵母細(xì)胞生長發(fā)育十分活躍，表現(xiàn)為細(xì)胞體積增大，基因轉(zhuǎn)錄和蛋白質(zhì)合成顯著增加。這些母源RNA和蛋白質(zhì)被儲存在生發(fā)泡(germinal vesicle，GV)和胞質(zhì)中，以支持卵母細(xì)胞減數(shù)分裂成熟和早期胚胎發(fā)育[58]。類似于原始卵泡卵母細(xì)胞，在應(yīng)對DNA損傷時，初級卵泡卵母細(xì)胞也會發(fā)生由TAp63介導(dǎo)的細(xì)胞凋亡反應(yīng)。基因敲除小鼠的初級卵泡卵母細(xì)胞能抵抗一定劑量的輻射而存活[42]。因此，在原始卵泡和初級卵泡中，DNA損傷的卵母細(xì)胞均傾向于通過TAp63介導(dǎo)的凋亡程序清除自身。然而，在初級卵泡之后的生長階段，卵母細(xì)胞中TAp63表達(dá)水平下降，因此生長階段的卵母細(xì)胞在發(fā)生DNA損傷后不易凋亡。Puy等[59]報道，0.5 Gy的射線誘發(fā)了小鼠原始卵泡的大量損失，而大量生長卵泡在經(jīng)8 Gy的射線照射后仍然存活。Luan等[40]也表明，與休眠的原始卵泡卵母細(xì)胞相比，激活后的卵母細(xì)胞對化療藥物(環(huán)磷酰胺)的敏感性更差。研究認(rèn)為，生長階段的卵母細(xì)胞更傾向于對DNA損傷進(jìn)行修復(fù)，此時卵母細(xì)胞中與DNA修復(fù)相關(guān)的基因表達(dá)增加[60～64]。對于DSBs損傷，與原始卵泡卵母細(xì)胞一樣，生長階段的卵母細(xì)胞也主要通過HR機制進(jìn)行修復(fù)[50]。然而，如果DNA損傷超過了一定的限度，卵母細(xì)胞也將發(fā)生凋亡。多柔比星(doxorubicin，DOX)和依托泊苷(etoposide，ETP)是廣泛使用的DNA損傷誘導(dǎo)藥物。低劑量DOX和ETP對小鼠卵泡的存活和生長影響較小，而高劑量DOX和ETP則會導(dǎo)致生長卵泡存活率大幅下降，卵母細(xì)胞凋亡率增加[65,66]。

2.3 完全生長的卵母細(xì)胞

卵泡生長發(fā)育的最后階段為成熟卵泡。成熟卵泡的卵母細(xì)胞是完全生長的卵母細(xì)胞。在完全生長的卵母細(xì)胞中，染色質(zhì)結(jié)構(gòu)已從非包圍核仁狀態(tài)轉(zhuǎn)變?yōu)榘鼑巳薁顟B(tài)，基因轉(zhuǎn)錄沉默[67]。在黃體生成素(luteinizing hormone，LH)峰的作用下，處于第一次減數(shù)分裂前期的完全生長的GV卵母細(xì)胞恢復(fù)減數(shù)分裂，發(fā)生生發(fā)泡破裂(germinal vesicle breakdown，GVBD)，進(jìn)入第一次減數(shù)分裂中期(metaphase I，MI)，此時通過交叉連接的同源染色體排列在赤道板上。隨后同源染色體在紡錘絲的牽引下分離并向兩極移動。移至兩極后，染色體解旋，核膜重建，同時細(xì)胞質(zhì)分裂，卵母細(xì)胞排出第一極體，卵母細(xì)胞中剩余每對同源染色體中的一條，至此第一次減數(shù)分裂完成。卵母細(xì)胞隨后進(jìn)入第二次減數(shù)分裂，并停滯在第二次減數(shù)分裂中期(metaphase II，MII)，此時通過著絲粒連接的高度螺旋化的姐妹染色體排列在赤道板上。以上的過程被稱為卵母細(xì)胞減數(shù)分裂成熟[58]。

轉(zhuǎn)錄沉默的卵母細(xì)胞的DNA修復(fù)依賴于細(xì)胞中儲存的相關(guān)mRNA和蛋白質(zhì)。在完全生長的GV和MII卵母細(xì)胞中，參與各種DNA修復(fù)途徑的mRNA和蛋白質(zhì)都已被確定[7,60～63,68,69]。研究已證明，完全生長的卵母細(xì)胞能夠通過HR和NHEJ機制對DSBs進(jìn)行修復(fù)[70]。在小鼠完全生長的GV卵母細(xì)胞中敲降、、等HR關(guān)鍵基因造成了卵母細(xì)胞DSBs增加，卵母細(xì)胞存活率降低[54]。蛋白磷酸酶4催化亞基基因(protein phosphatase 4 catalytic subunit，)是促進(jìn)HR修復(fù)的重要基因，條件性敲除小鼠的GV和MII卵母細(xì)胞的HR修復(fù)功能障礙，細(xì)胞出現(xiàn)大量DNA損傷[71]。Lee等[70]的研究進(jìn)一步表明，在減數(shù)分裂成熟期間，DSBs修復(fù)途徑從HR過渡到NHEJ，HR在GV卵母細(xì)胞中占優(yōu)勢，NHEJ在MII卵母細(xì)胞中占優(yōu)勢。此外，兩項研究也證明小鼠MII卵母細(xì)胞能夠通過NHEJ機制，而不是HR機制，對DSBs進(jìn)行修復(fù)[72,73]。原因可能是相比于長期停滯在第一次減數(shù)分裂前期雙線期的GV卵母細(xì)胞，一方面MII卵母細(xì)胞的染色體處于高度螺旋化的狀態(tài)，HR機制在細(xì)胞分裂中期發(fā)揮的作用受限，另一方面減數(shù)分裂成熟期間的卵母細(xì)胞可能不具備進(jìn)行HR修復(fù)所需的較長時間，因此能夠在整個細(xì)胞周期進(jìn)行快速修復(fù)的NHEJ機制是MII卵母細(xì)胞修復(fù)DSBs的主要選擇[74,75]。此外，BER途徑能夠修復(fù)氧化誘導(dǎo)的DNA損傷，即8-羥基脫氧鳥苷(8-hydroxy-2'-deoxyguanosine，8-OHdG)[76]。Lord等[77]發(fā)現(xiàn)，小鼠MII卵母細(xì)胞具有參與BER途徑的幾乎所有蛋白質(zhì)，并且卵母細(xì)胞受精后的一系列翻譯后修飾活動能夠激活BER途徑的關(guān)鍵蛋白，增強細(xì)胞的DNA修復(fù)能力。ICLs修復(fù)途徑的FANCE蛋白也能夠保護小鼠GV和MII卵母細(xì)胞的DNA[57]。以上研究共同表明，哺乳動物完全生長的卵母細(xì)胞中存在活躍的DNA修復(fù)活動。

3 DNA損傷對卵母細(xì)胞減數(shù)分裂成熟的影響及機制

前文已闡述，DNA損傷在一定程度上影響卵母細(xì)胞的存活和發(fā)育?？紤]到卵母細(xì)胞的功能，隨之而來的問題是，DNA損傷是否對卵母細(xì)胞的減數(shù)分裂成熟有所影響？眾所周知，卵母細(xì)胞長期處于第一次減數(shù)分裂的G2/前期。直到排卵時，卵母細(xì)胞恢復(fù)減數(shù)分裂，發(fā)育至MII期[58]。一些研究共同表明，外源誘導(dǎo)的DNA損傷不易導(dǎo)致GV卵母細(xì)胞停滯在G2/前期，大多數(shù)卵母細(xì)胞能夠恢復(fù)甚至完成第一次減數(shù)分裂。DNA損傷對卵母細(xì)胞的減數(shù)分裂成熟進(jìn)程影響較小，除非DNA損傷嚴(yán)重[6,70,78～82]。這與體細(xì)胞應(yīng)對DNA損傷時所發(fā)生的G2/M期停滯反應(yīng)有所不同。當(dāng)體細(xì)胞在G2/M期發(fā)生DNA損傷時，細(xì)胞啟動DDR，激活DNA損傷檢查點激酶，通過抑制CDC25磷酸酶最終抑制CDK1的激活，從而使細(xì)胞停滯在G2/M期，為修復(fù)病變提供時間[83]。卵母細(xì)胞不能建立強有力的G2/前期停滯反應(yīng)的原因可能是DNA損傷檢查點激酶被激活的強度不夠[78]。近期的兩項研究作出了更多的解釋。野生型P53誘導(dǎo)的磷酸酶1(wild-type p53-induced phosphatase 1，WIP1)能夠?qū)DR途徑的許多蛋白進(jìn)行去磷酸化[84]。Leem等[85]發(fā)現(xiàn)，在卵母細(xì)胞減數(shù)分裂成熟過程中持續(xù)表達(dá)的WIP1阻礙了ATM的磷酸化，從而抑制了DNA損傷檢查點的激活，細(xì)胞周期無法暫停。另一種解釋是小鼠卵母細(xì)胞的DNA損傷檢查點的激活是延遲的，在DNA損傷發(fā)生數(shù)小時(20 小時以上)后才啟動[86]。

在經(jīng)典的G2/M DNA損傷檢查點不能被有效激活的情況下，卵母細(xì)胞依然有能力誘導(dǎo)細(xì)胞周期停滯以應(yīng)對DNA損傷，這與紡錘體組裝檢查點(spindle assembly checkpoint，SAC)有關(guān)。SAC在紡錘體組裝過程中負(fù)責(zé)監(jiān)測著絲粒與微管的附著狀態(tài)，從而保障染色體的正確分離[87,88]。毒性藥物(如DOX、ETP)、紫外線B和電離輻射誘導(dǎo)的基因組損傷可激活SAC，誘導(dǎo)卵母細(xì)胞停滯在MI期。而抑制SAC則可以阻止DNA損傷誘導(dǎo)的MI期停滯反應(yīng)。然而，卵母細(xì)胞的基因組輕中度損傷可能不足以激活SAC，卵母細(xì)胞能夠發(fā)育至MII期。需注意的是，逃逸的卵母細(xì)胞存在DNA損傷、紡錘體變形、染色體紊亂等異常[70,78,80,87,88]。這些異?？赡軐β涯讣?xì)胞受精及后續(xù)胚胎發(fā)育過程造成影響。研究發(fā)現(xiàn)，有DNA損傷的小鼠MII卵母細(xì)胞受精后形成的胚胎發(fā)育異常，囊胚率顯著下降[71,72]。近期，Leem等[89]發(fā)現(xiàn)，在小鼠卵母細(xì)胞中存在一個依賴于DNA損傷檢查點1的介質(zhì)蛋白(mediator of DNA damage checkpoint 1，MDC1)的非經(jīng)典G2/M DNA損傷檢查點。在卵母細(xì)胞DNA損傷時，MDC1與后期促進(jìn)復(fù)合物/環(huán)狀體(anaphase promoting complex/cyclosome，APC/C)- APC/C激活蛋白CDH1(APC/C activator protein CDH1，CDH1)發(fā)生解離，游離的APC/C-CDH1介導(dǎo)了細(xì)胞周期蛋白B1(cyclin B1)降解從而推遲M期的進(jìn)入。

4 卵母細(xì)胞DNA損傷與修復(fù)相關(guān)研究的臨床價值

4.1 生殖衰老與抗氧化

當(dāng)今社會人們推遲生育的趨勢越來越明顯。隨著生育年齡的增長，女性的卵巢儲備和卵母細(xì)胞質(zhì)量大幅降低[90]。生殖衰老引起的卵母細(xì)胞質(zhì)量下降與卵母細(xì)胞的DNA損傷積累和DNA修復(fù)能力下降密切相關(guān)[1,91～95]。在人類、豬()、小鼠等多個物種中，隨著機體年齡增長，卵母細(xì)胞的DSBs損傷逐漸積累，關(guān)鍵的DNA修復(fù)蛋白如BRCA1、RAD51、ATM、卵母細(xì)胞表達(dá)蛋白(oocyte expressed protein，OOEP)等的表達(dá)水平也逐漸下降[54,96～98]。Horta等[99]發(fā)現(xiàn)與年輕小鼠相比，老年小鼠的卵母細(xì)胞修復(fù)精子DNA損傷的能力下降。在人類中也發(fā)現(xiàn)，高齡女性的卵母細(xì)胞在注射高精子DNA碎片指數(shù)的精子后會發(fā)育成質(zhì)量較差的胚胎，從而導(dǎo)致較低的植入率、懷孕率以及較高的流產(chǎn)率[100]。此外，全基因組關(guān)聯(lián)研究表明，基因組不穩(wěn)定性可能是絕經(jīng)發(fā)生的一個因素，進(jìn)一步提供了卵巢衰老與卵母細(xì)胞DNA修復(fù)能力密切相關(guān)的證據(jù)[93,101]。以衰老為例，機體內(nèi)源性因素誘導(dǎo)的DNA損傷在很大程度上影響了卵母細(xì)胞的質(zhì)量和發(fā)育能力。研究表明，誘導(dǎo)卵母細(xì)胞DNA損傷的內(nèi)源性因素主要是以ROS為代表的細(xì)胞內(nèi)代謝活動的產(chǎn)物[38,54,77,102,103]。因此，研究如何通過抗氧化方法減輕卵母細(xì)胞內(nèi)源性DNA損傷具有重要意義，有助于保護臨床中有需求人群的生育能力[91,104]。

目前研究已發(fā)現(xiàn)多種能夠減輕卵母細(xì)胞DNA損傷的抗氧化劑。褪黑素是一種能夠清除超氧陰離子和過氧化氫的強效抗氧化劑，能夠維持抗氧化代謝物的水平，減少ROS的積累。關(guān)于小鼠、大鼠()、豬等的研究發(fā)現(xiàn)，褪黑素能夠抑制卵母細(xì)胞ROS的產(chǎn)生，減輕化療藥物或衰老引起的DNA損傷，改善卵母細(xì)胞質(zhì)量[105～108]。褪黑素還能夠增強細(xì)胞的NHEJ修復(fù)活動，減輕DSBs損傷，促進(jìn)小鼠卵母細(xì)胞成熟[73]。輔酶Q10是一種天然抗氧化劑，能夠通過降低超氧化物和DNA損傷的水平來抑制氧化應(yīng)激和細(xì)胞凋亡，從而有效提高小鼠和大鼠的卵母細(xì)胞質(zhì)量[109,110]。N-乙酰-L-半胱氨酸是一種合成制劑，能夠刺激細(xì)胞合成谷胱甘肽。谷胱甘肽是由谷氨酸、半胱氨酸和甘氨酸組合而成的含巰基三肽，能夠通過巰基與氧自由基及其他親電體相互作用，也可作為各種酶的輔助因子來發(fā)揮抗氧化作用[111]。服用N-乙酰-L-半胱氨酸可以減輕小鼠卵母細(xì)胞的氧化DNA損傷，提高卵母細(xì)胞發(fā)育能力[112]。此外，煙酰胺單核苷酸(nicotinamide mononucleotide，NMN)和煙酰胺核糖苷(nicotinamide riboside，NR)也具有抗氧化功能。NMN和NR是生物合成煙酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide，NAD+)的必要前體。NAD+是一種重要的氧化還原輔助因子和酶底物，參與能量代謝和DNA修復(fù)等過程[113]。衰老和代謝異常引起機體NAD+水平下降。補充NMN和NR可降低ROS水平、減輕DNA損傷和抑制細(xì)胞凋亡，從而改善卵母細(xì)胞的發(fā)育潛力[114,115]。除使用藥物干預(yù)外，選擇健康的生活方式也是減輕機體氧化應(yīng)激的有效方法[116]。已有研究證明體育活動能顯著降低肥胖大鼠卵母細(xì)胞的NADPH氧化酶的活性，減少ROS的產(chǎn)生，減輕卵母細(xì)胞損傷，改善卵母細(xì)胞質(zhì)量[117,118]。

4.2 放化療患者的生育力保存

在臨床上，化療和放療是針對癌癥的常用治療手段。然而，在治療癌癥的同時，放療和化療對卵巢造成了嚴(yán)重的損害，直接損傷了卵母細(xì)胞DNA并誘導(dǎo)卵母細(xì)胞凋亡，進(jìn)而導(dǎo)致患者發(fā)生POI、不孕和提前絕經(jīng)等疾病[36,37]。對于接受放化療的有生育需求的女性來說，如何保存其生育力是值得研究且具有重要臨床意義的。目前人們提出了一些藥物治療的建議，包括性激素、鞘氨醇-1-磷酸、地塞米松等藥物。然而，沒有足夠的證據(jù)支持這些藥物的有效性和安全性[36,119]。更好的辦法是預(yù)先手術(shù)切除部分卵巢并冷凍保存，在癌癥治療結(jié)束后再將其重新植入患者體內(nèi)，然而這種方法有重新引入腫瘤細(xì)胞的風(fēng)險且受到手術(shù)時間的限制[120]。鑒于放化療誘導(dǎo)原始卵泡卵母細(xì)胞DNA損傷進(jìn)而導(dǎo)致卵母細(xì)胞凋亡的分子機制，靶向卵母細(xì)胞的DDR機制進(jìn)行藥物研發(fā)或許是保護患者生育力的有效策略。

抑制卵母細(xì)胞凋亡可能是保護放化療患者卵母細(xì)胞的潛在靶點之一。研究表示，通過敲除介導(dǎo)卵母細(xì)胞凋亡的基因，如、、等基因，小鼠的大量卵母細(xì)胞能夠在輻射后存活，并且小鼠能夠生育健康的子代[38,40,45,50,51]。通過對、、等基因敲除小鼠的卵母細(xì)胞進(jìn)行γH2AX(DSBs標(biāo)志物)免疫熒光染色，研究發(fā)現(xiàn)輻射誘導(dǎo)的卵母細(xì)胞的DSBs損傷在5天后基本消失。且在輻射發(fā)生后的24小時，基因敲除小鼠卵母細(xì)胞的RAD51蛋白水平明顯增加[38,50,51]。這說明，在人為阻止凋亡發(fā)生后，小鼠的卵母細(xì)胞激活了DNA修復(fù)途徑，對損傷的DNA進(jìn)行了修復(fù)。此外，Luan等[40]報道，在基因敲除小鼠的原始卵泡卵母細(xì)胞中，標(biāo)志線粒體活性的OPA1線粒體動力蛋白樣GTP酶(OPA1 mitochondrial dynamin like GTPase，OPA1)蛋白水平增加，標(biāo)志著卵母細(xì)胞生存能力增強。因此，抑制卵母細(xì)胞凋亡似乎可以間接促進(jìn)DNA修復(fù)和增強細(xì)胞生存能力。相較于基因編輯，使用抑制凋亡的小分子藥物是更可行的辦法。研究報道，靶向CHK2或ATM的小分子抑制劑可以阻止TAp63磷酸化，抑制輻射和化療藥物誘導(dǎo)的卵母細(xì)胞凋亡，對卵母細(xì)胞具有保護作用[40,45,121～124]。綜上所述，這些研究為放化療患者的生育力保護提供了理論依據(jù)和干預(yù)靶點，然而這些小分子制劑作為生育保護劑的潛力和安全性仍需進(jìn)一步研究。

此外，激活DNA修復(fù)途徑、補充關(guān)鍵的DNA修復(fù)因子等方法也顯現(xiàn)出了增強卵母細(xì)胞DNA修復(fù)能力的潛能。Sirtuins(SIRT)家族的許多成員是DNA修復(fù)途徑的關(guān)鍵因子[125]，SIRT1、SIRT6和SIRT7蛋白分別在HR、BER和NHEJ修復(fù)中發(fā)揮了重要作用[126～130]。因此，針對SIRT的靶向激活藥物可能具有改善卵母細(xì)胞質(zhì)量的潛能。白藜蘆醇是一種SIRT1激活劑。研究報道，在小鼠、豬、牛的卵母細(xì)胞體外培養(yǎng)基添加白藜蘆醇能夠減輕卵母細(xì)胞DNA損傷、改善卵母細(xì)胞質(zhì)量和提高早期胚胎發(fā)育能力[131～133]。此外，哺乳動物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信號通路的抑制劑雷帕霉素似乎也能夠增強細(xì)胞DNA修復(fù)能力。研究報道，在人類和小鼠的卵母細(xì)胞中，雷帕霉素能夠增強DDR相關(guān)基因的表達(dá)，減輕DNA損傷，提高卵母細(xì)胞的質(zhì)量和發(fā)育潛力[134～136]。此外，人們嘗試了對卵母細(xì)胞直接補充關(guān)鍵的DNA修復(fù)因子。Kujjo等[53]報道，向小鼠的老化卵母細(xì)胞中注射重組RAD51蛋白能夠減輕DSBs損傷，抑制卵母細(xì)胞凋亡，提高胚胎的發(fā)育能力。

5 結(jié)語與展望

與體細(xì)胞類似，卵母細(xì)胞也容易被各種因素誘發(fā)DNA損傷。在基礎(chǔ)研究和臨床治療中，輻射和化療藥物經(jīng)常造成卵母細(xì)胞DNA損傷和卵巢儲備損失。除了外源因素，卵母細(xì)胞也容易被ROS等內(nèi)源因素誘發(fā)DNA損傷。對于DNA損傷，卵母細(xì)胞能夠啟動DDR，發(fā)生包括DNA修復(fù)在內(nèi)的一系列活動。圖1總結(jié)了各發(fā)育階段卵母細(xì)胞的DDR與DNA修復(fù)機制。在卵母細(xì)胞中，有關(guān)DSBs損傷與修復(fù)的研究是最多的。研究已經(jīng)證明，各發(fā)育階段的卵母細(xì)胞均能通過HR或NHEJ機制修復(fù)DSBs損傷。然而其他DNA損傷類型的卵母細(xì)胞相關(guān)研究仍比較少。有趣的是，在應(yīng)對DNA損傷時，不同發(fā)育階段卵母細(xì)胞的DDR有所不同。原始卵泡卵母細(xì)胞和初級卵泡卵母細(xì)胞傾向于通過TAp63途徑啟動凋亡。然而，進(jìn)入生長階段后的卵母細(xì)胞更傾向于選擇修復(fù)，而不是凋亡，除非DNA損傷嚴(yán)重。卵母細(xì)胞這種選擇策略背后的原因不明。從進(jìn)化的角度看，在卵母細(xì)胞發(fā)育的最初階段，清除基因受損的卵母細(xì)胞能夠大大降低子代基因突變和染色體變異的風(fēng)險。此外，輕中度DNA損傷對卵母細(xì)胞的減數(shù)分裂成熟影響較小，而嚴(yán)重的DNA損傷則可以通過激活DNA損傷檢查點誘導(dǎo)卵母細(xì)胞G2/前期停滯，或者通過激活SAC誘導(dǎo)卵母細(xì)胞MI期停滯。需注意的是，即使逃避了細(xì)胞周期檢查點的卵母細(xì)胞能夠完成第一次減數(shù)分裂，然而DNA損傷、染色體變異等異?？赡艹掷m(xù)存在。這提示人們，在科研及臨床工作中，獲得的形態(tài)正常的MII卵母細(xì)胞可能存在DNA和染色體的異常，這些異?？赡軐β涯讣?xì)胞受精及早期胚胎發(fā)育造成影響。

生育高齡女性的卵巢儲備減少和卵母細(xì)胞質(zhì)量下降是生殖臨床中的常見現(xiàn)象。衰老引起卵母細(xì)胞DNA損傷積累和DNA修復(fù)能力下降，而ROS增多是衰老損傷細(xì)胞DNA的主要機制之一。研究表明，褪黑素等抗氧化劑能夠有效減輕ROS誘導(dǎo)的DNA損傷和提高卵母細(xì)胞發(fā)育能力。此外，在癌癥的臨床治療中，放化療容易造成卵母細(xì)胞DNA損傷和誘導(dǎo)卵母細(xì)胞凋亡，由此引起的POI對患者的生育能力造成了極大威脅。關(guān)于卵母細(xì)胞的DNA損傷與修復(fù)機制的研究似乎為此類患者保存生育力提供了新的策略。研究發(fā)現(xiàn)，通過基因編輯或小分子靶向制劑阻止卵母細(xì)胞凋亡可以間接促進(jìn)DNA修復(fù)，從而保護暴露于輻射和化療藥物的動物的生育能力。其次，向卵母細(xì)胞直接補充重要DNA修復(fù)因子以及使用小分子制劑激活DNA修復(fù)途徑等方法也初步在動物實驗中顯現(xiàn)出有效效果?？傮w而言，這些研究呈現(xiàn)出了較好的結(jié)果，然而涉及的藥物制劑的效果及作用機制仍需更多的評價和研究。

圖1 各發(fā)育階段卵母細(xì)胞的DDR與DNA修復(fù)機制

DNA損傷發(fā)生時，原始卵泡和初級卵泡的卵母細(xì)胞均通過TAp63介導(dǎo)的反應(yīng)發(fā)生凋亡。卵母細(xì)胞進(jìn)入生長階段后，TAp63水平下降，參與MMR、BER、NER、HR和NHEJ等DNA修復(fù)途徑的基因表達(dá)水平增加。HR和NHEJ機制在卵母細(xì)胞的DSBs修復(fù)中發(fā)揮重要作用。在卵母細(xì)胞減數(shù)分裂成熟期間，DSBs修復(fù)途徑從HR過渡到NHEJ。對于ICLs和8-OHdG損傷，卵母細(xì)胞可分別通過FANCE和BER途徑進(jìn)行修復(fù)。卵母細(xì)胞的其他類型DNA損傷的修復(fù)機制有待驗證。卵母細(xì)胞缺乏經(jīng)典的由ATM-CHK1介導(dǎo)的G2/前期DNA損傷檢查點，但似乎具有一個由MDC1介導(dǎo)的非經(jīng)典G2/前期DNA損傷檢查點。此外，SAC介導(dǎo)了卵母細(xì)胞的MI期停滯反應(yīng)。

綜上所述，卵母細(xì)胞的DDR與DNA修復(fù)機制十分復(fù)雜，許多問題迄今只在動物研究中得到了初步回答，關(guān)于人類的相關(guān)研究仍很缺乏。伴隨著高通量技術(shù)、基因編輯技術(shù)、實驗技術(shù)等的快速發(fā)展，人們將不斷拓寬和加深對于哺乳動物卵母細(xì)胞的DNA損傷與修復(fù)機制的認(rèn)識。此領(lǐng)域的研究將為開發(fā)生育力保護策略提供理論基礎(chǔ)和新的方向，具有重要意義。

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Advances in the study of DNA damage and repair in mammalian oocytes

Nan Zhang1, Jue Zhang2, Ge Lin1,2

DNA damage is one of the key factors affecting gametogenesis and embryo development. Oocytes are susceptible to DNA damage induced by various endogenous and exogenous factors (e.g., reactive oxygen species, radiation, chemotherapeutic agents, etc.). Current research has revealed that oocytes at various developmental stages are able to respond to various types of DNA damage, repairing DNA or initiating apoptosis through complex mechanisms. Primordial follicular oocytes are more susceptible to apoptosis induced by DNA damage than oocytes entering the growth stage. DNA damage is less likely to induce arrest of the meiotic maturation process in oocytes, however the developmental capacity of oocytes carrying DNA damage is significantly reduced. In clinical practice, aging, radiation and chemotherapy are common causes of oocyte DNA damage, reduced ovarian reserve and infertility in women. Therefore, various methods that can reduce DNA damage and enhance DNA repair in oocytes have been tried in an attempt to protect oocytes. In this review, we systematically summarize the mechanisms of DNA damage and repair in mammalian oocytes at various developmental stages and discuss their potential clinical value with the aim to provide new strategies for fertility protection.

oocytes; DNA damage; DNA repair; DNA damage response

2023-02-02;

2023-03-23;

2023-03-29

中國博士后科學(xué)基金(編號：2021M690983)資助[Supported by the Fellowship of China Postdoctoral Science Foundation (No. 2021M690983)]

張楠，在讀碩士研究生，專業(yè)方向：生殖醫(yī)學(xué)。E-mail: csudnn@csu.edu.cn

林戈，博士，研究員，研究方向：生殖醫(yī)學(xué)。E-mail: linggf@hotmail.com

10.16288/j.yczz.23-018

(責(zé)任編委: 黃俊)