泛素-蛋白酶體途徑在生殖細(xì)胞減數(shù)分裂過(guò)程中的作用及機(jī)制

彭馥芝 董蓮花 冉茂良 翁 波 陳 斌*

(1.湖南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，長(zhǎng)沙 410128；2.畜禽遺傳改良湖南省重點(diǎn)實(shí)驗(yàn)室，長(zhǎng)沙 410128)

泛素-蛋白酶體途徑(ubiquitin-proteasome pathway，UPP)通過(guò)三酶級(jí)聯(lián)將泛素連接于靶蛋白以使其泛素化，為介導(dǎo)真核生物蛋白質(zhì)降解調(diào)節(jié)的主要途徑。此外，泛素化在蛋白質(zhì)穩(wěn)定性[1]、蛋白質(zhì)運(yùn)輸[2]、細(xì)胞分化[3]、細(xì)胞周期進(jìn)程[4]等過(guò)程中發(fā)揮重要作用，與脂質(zhì)代謝[5]、肌肉發(fā)育[6]、神經(jīng)元形態(tài)發(fā)生[7]等生理過(guò)程相關(guān)，UPP異常則導(dǎo)致肌肉萎縮[8]、炎癥反應(yīng)[9]、睪丸腫瘤[10]等病變發(fā)生。研究證實(shí)，泛素相關(guān)組件及去泛素酶在動(dòng)物配子生成中普遍表達(dá)，在減數(shù)分裂過(guò)程中的減數(shù)分裂同源重組[11]、減數(shù)分裂性染色體失活[12]、卵母細(xì)胞減數(shù)分裂恢復(fù)[13]、第一極體(the first polar body，PBI)排出[14]及精卵融合[15]等過(guò)程中發(fā)揮著重要作用，UPP的紊亂或UPP組件的突變將導(dǎo)致這些生物過(guò)程發(fā)生損傷及配子發(fā)育缺陷。例如，初級(jí)精母細(xì)胞粗線(xiàn)期染色體聯(lián)會(huì)重組，組蛋白多聚泛素化，人工敲除組蛋白泛素化出現(xiàn)減數(shù)分裂中斷并最終導(dǎo)致不育[16]；圓形精子細(xì)胞中，組蛋白泛素化有助于魚(yú)精蛋白替換[17]；卵母細(xì)胞缺失Cullin4(CUL4)導(dǎo)致減數(shù)第1次分裂恢復(fù)延遲[18]；敲減泛素結(jié)合酶2C(UBE2C)導(dǎo)致第一極體排出受阻及染色體分離紊亂[19]。研究UPP在動(dòng)物配子生成中的生物學(xué)功能及作用機(jī)制，不但能推進(jìn)對(duì)生殖過(guò)程的進(jìn)一步了解，對(duì)治療人類(lèi)不孕不育及提高經(jīng)濟(jì)動(dòng)物繁殖性能也具有重要意義。本文綜述了泛素-蛋白酶體通路在動(dòng)物生殖細(xì)胞減數(shù)分裂過(guò)程及配子生成中的信號(hào)傳導(dǎo)及調(diào)節(jié)機(jī)制，以期為后續(xù)的相關(guān)研究提供參考。

1 UPP概述

UPP由泛素、泛素激活酶(ubiquitin-activating enzyme，E1)、泛素結(jié)合酶(ubiquitin-conjugating enzyme，E2)、泛素-蛋白質(zhì)連接酶(ubiquitin-protein ligase，E3)及蛋白酶體(proteasomes)組成。泛素分子在3種酶的共同作用下與底物蛋白共價(jià)結(jié)合的過(guò)程稱(chēng)為泛素化[20]，其中多聚泛素化在蛋白質(zhì)降解及信號(hào)轉(zhuǎn)導(dǎo)過(guò)程中發(fā)揮重要作用。泛素分子含7個(gè)賴(lài)氨酸殘基(K6、 K11、 K27、 K29、K33、K48及 K63)，每個(gè)殘基都能與泛素連接，從而形成至少7種不同的多聚泛素連接[21]。泛素化受三酶級(jí)聯(lián)反應(yīng)調(diào)控。E1以ATP依賴(lài)性激活泛素后，其硫基與泛素C端的羧基連接形成硫酯鍵；隨后通過(guò)交酯化過(guò)程將活化泛素從E1轉(zhuǎn)移至E2，形成E2-泛素中間物；最后E3募集底物蛋白并與E2-泛素中間物結(jié)合，催化泛素轉(zhuǎn)移至底物蛋白的賴(lài)氨酸殘基上，從而形成泛素異肽鏈[22]。靶蛋白在3種酶的作用下共價(jià)連接幾個(gè)泛素分子，被26S蛋白酶體識(shí)別后水解[22]。UPP調(diào)控不同信號(hào)通路蛋白質(zhì)的表達(dá)，從而在不同生物過(guò)程中發(fā)揮作用。UPP組件在生殖細(xì)胞發(fā)育各階段表達(dá)，在調(diào)控減數(shù)分裂及雌雄配子生成方面具有重要作用。

2 UPP與雌性生殖細(xì)胞減數(shù)分裂

2.1 卵母細(xì)胞減數(shù)第1次分裂恢復(fù)

哺乳動(dòng)物胚胎期卵原細(xì)胞分化為初級(jí)卵母細(xì)胞后，細(xì)胞周期休止于減數(shù)第1次分裂(meiosis Ⅰ，M Ⅰ)前期，直到進(jìn)入青春期后，卵母細(xì)胞恢復(fù)減數(shù)分裂進(jìn)行細(xì)胞發(fā)育形成具有受精能力的卵子。研究證實(shí)，后期促進(jìn)復(fù)合物/細(xì)胞周期體(anaphase-promoting complex/cyclosome，APC/C)在卵母細(xì)胞M Ⅰ恢復(fù)中具有重要作用。APC/C為Cullin-RING連接酶(Cullin-RING ligases，CRL)，APC/C通過(guò)與適配蛋白——細(xì)胞分裂周期蛋白20同源蛋白1(Cdc20 homolog 1，Cdh1)和細(xì)胞分裂周期蛋白20(cell division cycle protein 20，Cdc20)結(jié)合，形成APC/CCdh1或APC/CCdc20復(fù)合體，從而發(fā)揮E3活性，其中Cdh1和Cdc20與底物結(jié)合并將其呈遞至APC/C[23]。細(xì)胞周期蛋白Cyclin B和分離酶抑制蛋白(securin)為APC/C主要底物，卵母細(xì)胞減數(shù)分裂休止期必須避免Cyclin B和securin的過(guò)早積累。青春期時(shí)，卵母細(xì)胞恢復(fù)減數(shù)分裂，該階段卵母細(xì)胞Cyclin B積累并通過(guò)激酶/磷酸酶激活Cyclin B與細(xì)胞周期蛋白依賴(lài)性激酶1(cyclin-dependent kinase 1，Cdk1)構(gòu)成的復(fù)合物——促成熟因子(maturation promoting factor，MPF)，進(jìn)而使卵母細(xì)胞核膜破裂并退出M Ⅰ前期，而這一過(guò)程與APC/CCdh1的微調(diào)作用密切相關(guān)[13,24]。研究顯示，APC/CCdh1為哺乳動(dòng)物卵母細(xì)胞M Ⅰ前期和前中期的主要形式，APC/CCdh1活性對(duì)于減緩M Ⅰ前期Cyclin B積累及維持securin和Cyclin B平衡必不可少[25]，進(jìn)而在維持卵母細(xì)胞減數(shù)分裂休止中發(fā)揮重要作用[26]。此外，研究顯示，屬于E2的UBE2C、泛素結(jié)合酶2S(UBE2S)、泛素結(jié)合酶2D(UBE2D)調(diào)節(jié)小鼠卵母細(xì)胞減數(shù)分裂中APC/C活性，促進(jìn)卵母細(xì)胞染色體分離，并與紡錘體的形成相關(guān)。這些酶的缺失導(dǎo)致M Ⅰ胞質(zhì)分裂水平降低50%，而其過(guò)表達(dá)導(dǎo)致胞質(zhì)分裂速率提高2倍，高活性的UBE2C導(dǎo)致M Ⅰ的恢復(fù)[27]。

Cullin蛋白家族成員CUL4在卵母細(xì)胞M Ⅰ過(guò)程中也發(fā)揮著重要生理作用。Cullin蛋白內(nèi)無(wú)完整的RING結(jié)構(gòu)域，但其C端可通過(guò)結(jié)合RING家族的小蛋白如RBX1/2(ROC1/2)等構(gòu)成CRL類(lèi)E3復(fù)合物，負(fù)責(zé)與E2結(jié)合，Cullin蛋白N端則通過(guò)橋接蛋白與不同底物識(shí)別亞基結(jié)合，從而負(fù)責(zé)不同底物蛋白的募集，進(jìn)而在不同生物學(xué)過(guò)程中發(fā)揮作用[28]。CUL4的C端及N端分別與RBX1及橋接蛋白——DNA損傷結(jié)合蛋白1(damaged DNA binding protein 1，DDB1)結(jié)合，構(gòu)成DDB1-CUL4-RBX1 E3復(fù)合物[29]，DDB1通過(guò)結(jié)合含WD40重復(fù)結(jié)構(gòu)域的底物識(shí)別亞基DCAF1(DDB1-CUL4 associated factor 1)/VPRBP，進(jìn)而結(jié)合并降解特異底物。研究顯示，DCAF1能識(shí)別蛋白磷酸酶2A亞基(PP2A-A)，使其聚泛素化并靶向蛋白酶體降解。作為細(xì)胞周期調(diào)節(jié)因子，PP2A-A在卵母細(xì)胞減數(shù)分裂過(guò)程中發(fā)揮作用[18]，PP2A-A為維持MⅠ前期生發(fā)泡(germinal vesicle，GV)期卵母細(xì)胞正常休止所必需，生發(fā)泡破裂(germinal vesicle break down，GVBD)期卵母細(xì)胞PP2A-A蛋白水平開(kāi)始降低，小鼠卵母細(xì)胞特異性缺失DDB1或DCAF1會(huì)發(fā)生PP2A-A的積累及減數(shù)第1次分裂恢復(fù)的延遲，CRL4缺失也會(huì)使卵母細(xì)胞同源染色體分離受到抑制[18]。

2.2 第一極體排出

卵母細(xì)胞M Ⅰ完成的標(biāo)志為第一極體的排出，APC/CCdc20活性與該過(guò)程密切相關(guān)。研究顯示，APC/CCdh1在M Ⅰ前期和前中期先被激活，而M Ⅰ后期Cdk1介導(dǎo)激活的APC/CCdc20則為主要活性形式，APC/CCdc20靶向securin和Cyclin B1降解，進(jìn)而調(diào)控染色體的濃縮分離及第一極體的排出[14,30]。Cdk1蛋白酶抑制劑導(dǎo)致卵母細(xì)胞第1次分裂Cdk1失活及第一極體排出，而去除抑制劑的卵母細(xì)胞不能進(jìn)入減數(shù)第2次分裂(meiosis Ⅱ，M Ⅱ)。紡錘體組裝檢查點(diǎn)(spindle assembly checkpoint，SAC)監(jiān)管機(jī)制使染色體著絲粒正確附著于微管，也是調(diào)節(jié)APC/CCdc20活性的機(jī)制。在中期之前，SAC保持活性，并可能通過(guò)SAC組件——有絲分裂阻滯缺陷蛋白2(mitotic arrest deficient 2，Mad2)與Cdc20結(jié)合，阻礙APC/C與Cdc20之間互作，從而抑制APC/CCdc20活性[31]。當(dāng)所有染色體正確連接時(shí)，SAC關(guān)閉而APC/CCdc20活性達(dá)到峰值，APC/CCdc20通過(guò)降解Cyclin B1和securin，分離酶得以切開(kāi)連接染色體的環(huán)狀黏連蛋白(cohesin)促使染色體分離，對(duì)隨后的第一極體的排出具有關(guān)鍵作用。此外，SCFβ-TrCP-EMI1-APPC/C通路在小鼠卵母細(xì)胞MⅠ進(jìn)程及第一極體排出中同樣具有重要作用。與DDB1-CUL4-RBX1泛素連接酶復(fù)合體類(lèi)似，SCF(Skp1-Cullin-F-box)同為CRL類(lèi)E3，不同的是SCF的支架蛋白為Cullin1，Cullin1的C端同樣與RBX1結(jié)合，N端則以Skp1為橋接蛋白與F-box蛋白連接，進(jìn)而識(shí)別特異性底物。SCF泛素連接酶的底物——有絲分裂早期抑制蛋白1(early mitotic inhibitor-1,Emi1)為APC/C抑制劑，能抑制APC/C活性。SCF重要組成部分——RBX1在卵母細(xì)胞成熟過(guò)程中圍繞并隨著紡錘體和濃縮染色體遷移，用相應(yīng)的小干擾RNA(siRNA)敲低Rbx1導(dǎo)致卵母細(xì)胞第一極體排出率降低且大多數(shù)細(xì)胞阻滯于M Ⅰ中期，下調(diào)Rbx1的表達(dá)也導(dǎo)致Emi1的積累以及securin和Cyclin B1的表達(dá)明顯增加[32]。

泛素連接形式及蛋白酶體活性對(duì)第一極體的排出同樣具有重要影響。研究發(fā)現(xiàn)K-11泛素連接為第一極體排出的必要信號(hào)[19,29]。通過(guò)對(duì)小鼠GV期卵母細(xì)胞顯微注射泛素突變體以阻斷泛素鏈的延伸的試驗(yàn)研究發(fā)現(xiàn)，卵母細(xì)胞注射被精氨酸取代的K-11突變泛素分子，其第一極體的排出被顯著干擾，染色體分離過(guò)程也嚴(yán)重受損[19]。相較于野生型泛素注射控制組，微注射K-11突變多聚泛素鏈組卵母細(xì)胞出現(xiàn)染色體分離失敗及第一極體排出顯著減少[29]。蛋白酶體的催化活性對(duì)于MPF活性降低及第一極體的排出是必不可少。卵母細(xì)胞恢復(fù)減數(shù)分裂后，蛋白酶體轉(zhuǎn)移到紡錘體，蛋白酶體抑制劑——MG132處理大鼠卵母細(xì)胞出現(xiàn)cyclin B積累及MPF活性增強(qiáng)，且細(xì)胞阻遏于M Ⅰ中期，阻礙第一極體的排出[33]。此外，敲減UBE2C、UBE2S及UBE2D中任一E2，均顯示出第一極體排出障礙、紡錘體形成及染色體分離紊亂[19]。但APC/C在哺乳動(dòng)物雄性生殖細(xì)胞減數(shù)分裂中的作用機(jī)制還有待進(jìn)一步研究。

綜述所述，UPP在卵子發(fā)生中的作用見(jiàn)圖1。

3 UPP與雄性生殖細(xì)胞減數(shù)分裂

3.1 性染色體失活

哺乳動(dòng)物雄性生殖細(xì)胞減數(shù)分裂過(guò)程中，M I前期精母細(xì)胞X和Y染色體于擬常染色體區(qū)聯(lián)會(huì)濃縮形成XY小體(XY body)，XY小體具有轉(zhuǎn)錄沉默現(xiàn)象，稱(chēng)為減數(shù)分裂性染色體失活(meiotic sex chromosome inactivation，MSCI)[34]，該過(guò)程與減數(shù)分裂進(jìn)程密切相關(guān)，且能阻遏抑制精子發(fā)生基因的表達(dá)，對(duì)精子發(fā)生至關(guān)重要[35]。XY小體富含泛素化組蛋白H2A(uH2A)，其水平在精母細(xì)胞粗線(xiàn)期達(dá)到峰值，為性染色體轉(zhuǎn)錄沉默標(biāo)志[36]。研究顯示，果蠅及哺乳動(dòng)物中uH2A與基因阻遏相關(guān)[37]，對(duì)MSCI可能具有重要意義。屬于E3的UBR2與屬于E2的HR6B互作，介導(dǎo)H2A泛素化，UBR2缺失小鼠M Ⅰ精母細(xì)胞表現(xiàn)出H2A泛素化及MSCI缺失，而MSCI缺失可能激活粗線(xiàn)期檢查點(diǎn)(check point)機(jī)制并導(dǎo)致M Ⅰ阻滯[38]。

MSCI另一顯著標(biāo)志為組蛋白H3第4位賴(lài)氨酸(H3K4)二甲基化降低[39]，這一過(guò)程由HR6B與另一屬于E3的RAD18互作介導(dǎo)。HR6B或RAD18功能缺失導(dǎo)致M Ⅰ雙線(xiàn)期X、Y染色體H3K4二甲基化水平增加及沉默基因去阻遏現(xiàn)象的發(fā)生[40]。研究顯示，RAD18通過(guò)結(jié)合重組酶RAD51C介導(dǎo)DNA損傷后的同源重組修復(fù)，Rad18沉默小鼠的精母細(xì)胞M Ⅰ粗線(xiàn)期X、Y染色體聯(lián)會(huì)失敗、H3K4二甲基化增加及相應(yīng)的X連鎖基因去阻遏，同時(shí)小鼠表現(xiàn)出生育力低下、體重降低及睪丸體積減少[41]。研究證實(shí)，屬于E3的環(huán)指蛋白8(RING finger protein 8，RNF8)通過(guò)磷酸激酶ATM(ataxia telangiectasia mutated kinase)途徑介導(dǎo)H2AX(H2A的變體)第139位絲氨酸的磷酸化(γ-H2AX)[42]，Rnf8基因缺失小鼠性染色體出現(xiàn)正常的γ-H2AX的積聚及MSCI的啟動(dòng)，但XY小體不發(fā)生泛素化[13]。此外，泛素連接酶Ret finger蛋白(RFP)為轉(zhuǎn)錄抑制因子，能與核基質(zhì)結(jié)合蛋白及雙鏈DNA相互作用，于精母細(xì)胞M Ⅰ期性染色體異步聯(lián)會(huì)至關(guān)重要[43]。

3.2 減數(shù)分裂進(jìn)程

UPP組件中屬于E1的UBA6為減數(shù)分裂引發(fā)因子，與其他組織相比，人和小鼠UBA6 mRNA及蛋白表達(dá)水平在睪丸組織中最高。除了在新生小鼠生殖細(xì)胞表達(dá)，UBA6在PND10(postnatal day)的精原細(xì)胞和M Ⅰ前細(xì)線(xiàn)期精母細(xì)胞細(xì)胞質(zhì)中表達(dá)最高，可能在啟動(dòng)M Ⅰ方面發(fā)揮作用[44]，而PND20時(shí)則在精母細(xì)胞M Ⅰ細(xì)線(xiàn)期和偶線(xiàn)期細(xì)胞核中表達(dá)。除了參與卵母細(xì)胞M Ⅰ及第一極體的排出過(guò)程，屬于E3的SCFβ-TrCP也參與雄性生殖細(xì)胞減數(shù)分裂。β-轉(zhuǎn)導(dǎo)重復(fù)相容蛋白(beta-transducin repeats-containing proteins，β-TrCP)為Skp1-Cullin-F-box泛素連接酶復(fù)合物結(jié)構(gòu)中的F-box蛋白，β-TrCP缺失小鼠出現(xiàn)M Ⅰ中期精母細(xì)胞生成增加、精子細(xì)胞生成減少。β-TrCP缺失小鼠睪丸表現(xiàn)出SCFβ-TrCP底物——Emi1、細(xì)胞周期蛋白Cyclin A等細(xì)胞周期調(diào)節(jié)因子水平的增加，可能為其導(dǎo)致減數(shù)分裂缺陷的分子機(jī)制[45]。此外，維持睪丸自由泛素單體平衡重要基因——聚泛素基因Ubi-p63E，于果蠅精母細(xì)胞正常染色體凝聚及M Ⅰ中G2期向M期轉(zhuǎn)變至關(guān)重要，Ubi-p63E突變導(dǎo)致M Ⅰ期阻滯[46]。

M Ⅰ：減數(shù)第1次分裂 meiosis Ⅰ；M Ⅱ：減數(shù)第2次分裂 meiosis Ⅱ；PBI：第一極體 the first polar body；E2：泛素結(jié)合酶 ubiquitin-conjugating enzyme；E3：泛素-蛋白質(zhì)連接酶 ubiquitin-protein ligase；UBE2C：泛素結(jié)合酶2C ubiquitin-conjugating enzyme 2C；UBE2S：泛素結(jié)合酶2S ubiquitin-conjugating enzyme 2S；UBE2D：泛素結(jié)合酶2D ubiquitin-conjugating enzyme 2D；APC/C：后期促進(jìn)復(fù)合物/細(xì)胞周期體 anaphase-promoting complex/cyclosome；Cdc20：細(xì)胞分裂周期蛋白20 cell division cycle protein 20；Cdh1：Cdc20同源蛋白1 CDC20 homolog 1；CRL4：Cullin-RING連接酶4 Cullin-RING ligase 4；Mad2：有絲分裂阻滯缺陷蛋白2 mitotic arrest deficient 2； PP2A-A：蛋白磷酸酶2A亞基 protein phosphatase 2A。

“↑”表示表達(dá)量升高，“↓”表示表達(dá)量降低。

“↑” represented the increase of expression, and the “↓” represented the decrease of expression.

圖1UPP在卵子發(fā)生中的作用

Fig.1 The role of UPP during oogenesis[14,18,23-27,30-31]

屬于E3的CUL4在雄性生殖細(xì)胞減數(shù)分裂進(jìn)程中同樣具有重要生理作用。RNA干擾失活秀麗隱桿線(xiàn)蟲(chóng)CUL4，導(dǎo)致DNA復(fù)制起始蛋白CDT-1降解失敗?；虼虬行∈驝ul4導(dǎo)致CDT-1、磷酸化p53和錯(cuò)配修復(fù)蛋白MLH1積聚增加及M Ⅱ粗線(xiàn)期及雙線(xiàn)期細(xì)胞死亡率增加[29]。盡管有正常的聯(lián)會(huì)復(fù)合體及DNA雙鏈斷裂(DNA double-strand breaks，DSBs)修復(fù)，但小鼠M Ⅱ雙線(xiàn)期精母細(xì)胞重組小結(jié)MLH1解聚發(fā)生延遲，可能為導(dǎo)致M Ⅱ發(fā)生中斷的分子機(jī)制。中華絨螯蟹(Eriocheirsinensis)CUL4、細(xì)胞增殖核抗原(proliferating cell nuclear antigen，PCNA)及細(xì)胞周期蛋白p21、p27、p53在初級(jí)精母細(xì)胞階段具有高轉(zhuǎn)錄水平，在精子生成階段則水平降低(除p27之外)，p53介導(dǎo)的生殖細(xì)胞自發(fā)凋亡可能為消除生殖細(xì)胞缺陷的質(zhì)量監(jiān)控機(jī)制，而CRL4復(fù)合物可能通過(guò)維持初級(jí)精母細(xì)胞內(nèi)p53、p21及p27平衡進(jìn)而調(diào)控雄性生殖細(xì)胞M Ⅱ進(jìn)程[47]。

綜上所述，UPP在精子發(fā)生中的作用見(jiàn)圖2。

4 UPP與生殖細(xì)胞減數(shù)分裂重組

DSBs及DSBs修復(fù)為M Ⅰ前期重要事件，同源重組(homologous recombination，HR)為DSBs修復(fù)途徑之一，減數(shù)分裂重組是確保M Ⅰ前期生殖細(xì)胞遺傳物質(zhì)高效交換及保持基因組完整性的關(guān)鍵步驟。研究顯示，酵母中屬于E2的RAD6與屬于E3的BRE1互作調(diào)控組蛋白H2B的K123位發(fā)生單泛素化[48]。RAD6突變導(dǎo)致孢子生成及DNA修復(fù)缺陷[49]，失活BRE1或突變H2B殘基導(dǎo)致減數(shù)分裂中DSBs的減少，H2B的K123位發(fā)生突變也導(dǎo)致細(xì)胞分裂停滯于M Ⅰ期，表明RAD6/BRE1調(diào)控的H2B單泛素化為DSBs形成機(jī)制[50]。哺乳動(dòng)物中屬于E2的HR6A及HR6B(與酵母RAD6同源)介導(dǎo)DNA損傷修復(fù)[51]。Hr6b基因敲除小鼠不育，在第一精子發(fā)生波中表現(xiàn)出初級(jí)精母細(xì)胞凋亡增加、M Ⅰ粗線(xiàn)期聯(lián)會(huì)復(fù)合體較長(zhǎng)、近端粒區(qū)出現(xiàn)聯(lián)會(huì)復(fù)合體蛋白缺失及MLH1含量的增加，表明HR6B可能具有抑制減數(shù)分裂重組的作用[52-53]。研究表明，HR6A/HR6B與屬于E3的UBR2互作[54]，在維持基因組完整性及同源重組修復(fù)(homologous recombination repair，HRR)雙鍵斷裂中具有重要作用[55]。Ubr2基因缺失小鼠睪丸細(xì)胞缺乏完整的聯(lián)會(huì)復(fù)合體(synaptonemal complex)，造成細(xì)胞凋亡，進(jìn)而導(dǎo)致小鼠不育。此外，Ubr2基因單核苷酸多態(tài)性分析表明其與男性非梗阻性無(wú)精子癥相關(guān)[56]。

M Ⅰ：減數(shù)第1次分裂 meiosis Ⅰ；M Ⅱ：減數(shù)第2次分裂 meiosis Ⅱ；E1：泛素激活酶 ubiquitin-activating enzyme；E2：泛素結(jié)合酶 ubiquitin-conjugating enzyme；E3：泛素-蛋白質(zhì)連接酶 ubiquitin-protein ligase；β-TrCP：β-轉(zhuǎn)導(dǎo)重復(fù)相容蛋白 beta-transducin repeats-containing proteins；MDC1：mediator of DNA damage checkpoint 1；Emi1：有絲分裂早期抑制蛋白1 early mitotic inhibitor-1；DSBs：DNA雙鏈斷裂 DNA double-strand breaks；MSCI：減數(shù)分裂性染色體失活 meiotic sex chromosome inactivation；CUL4A：Cullin4A；SCF：Skp1-Cullin-F-box；RNF4：環(huán)指蛋白4 RING finger protein 4；RNF8：環(huán)指蛋白8 RING finger protein 8；RNF168：環(huán)指蛋白168 RING finger protein 168。

圖2UPP在精子發(fā)生中的作用

Fig.2 The role of UPP during spermatogenesis[11,16,29,38-41,44-45,47-48]

DNA雙鍵斷裂后，γ-H2AX被標(biāo)記到DSBs損傷位點(diǎn)，其磷酸化的第139位絲氨酸與MDC1(mediator of DNA damage checkpoint 1)蛋白作用并將MDC1招募到損傷位點(diǎn)，進(jìn)而激活RNF8-RNF168介導(dǎo)的泛素化通路[57]。屬于E3的RNF8和RNF168與屬于E2的UBC13互作，催化損傷染色體的組蛋白H2A及H2AX[58]發(fā)生K-63連接的多聚泛素化，進(jìn)而促進(jìn)DNA損傷應(yīng)答蛋白——P53結(jié)合蛋白(P53 binding protein 1，53BP1)和乳腺癌易感蛋白1(breast cancer susceptibility gene 1，BRCA1)對(duì)損傷位點(diǎn)的識(shí)別與結(jié)合[59]，BRCA1 N端的環(huán)指結(jié)構(gòu)域與BRCA1相關(guān)環(huán)狀蛋白(BRCA1-associated RING domain，BARD1)結(jié)合形成的異源二聚體——BRCA1/BARD1具有E3活性，通過(guò)與屬于E2的UBCH5C互作催化K-6連接的多聚泛素鏈合成，介導(dǎo)DSBs修復(fù)[60]。然而，有研究顯示，環(huán)指蛋白R(shí)NF169與53BP1和BRCA1競(jìng)爭(zhēng)性結(jié)合uH2A，負(fù)反饋調(diào)節(jié)同源重組修復(fù)[61]。而SUMO依賴(lài)的E3(SUMO-dependent ubiquitin ligase)RNF4能泛素化SUMO化修飾的DNA損傷檢查點(diǎn)蛋白1(mediator of DNA damage checkpoint protein 1，MDC1)及BRCA1，與同源重組修復(fù)蛋白R(shí)AD51互作參與DSBs修復(fù)[11]，RNF4缺失小鼠精母細(xì)胞凋亡增加并出現(xiàn)精子發(fā)生缺陷，小鼠胚胎成纖維細(xì)胞Rnf4hypo/hypo電離輻射后發(fā)生DNA永久性損傷[11]。

CUL4在生殖細(xì)胞減數(shù)分裂重組中也發(fā)揮重要作用。CUL4的C端與含環(huán)指結(jié)構(gòu)域小蛋白R(shí)OC1/RNF75結(jié)合，其N(xiāo)端則與DNA損傷結(jié)合蛋白1(damaged DNA binding protein 1，DDB1)結(jié)合構(gòu)成DDB1-CUL4-ROC1 E3復(fù)合物，其中DDB1負(fù)責(zé)特定靶蛋白的募集，而ROC1則與E2結(jié)合，介導(dǎo)靶蛋白泛素化[62]。哺乳動(dòng)物含2種Cul4基因：Cul4A(常染色體)和Cul4B(X染色體)[16]。研究表明，Cul4A截?cái)啾磉_(dá)小鼠不發(fā)生ROC1連接而表現(xiàn)出不育；Cul4A-/-第4～8外顯子缺失未檢測(cè)到截短蛋白，粗線(xiàn)期精母細(xì)胞發(fā)生永久性DSBs，出現(xiàn)同源重組缺陷，雄性小鼠睪丸細(xì)胞出現(xiàn)顯著的凋亡及M Ⅰ前期初級(jí)精母細(xì)胞缺乏，表現(xiàn)出嚴(yán)重的精子發(fā)生障礙及不育[16]。

5 去泛素酶(deubiquitinating enzymes，DUBs)在減數(shù)分裂中的作用

DUBs通過(guò)水解底物蛋白上的多聚泛素鏈逆轉(zhuǎn)蛋白泛素化，以維持蛋白質(zhì)代謝平衡。研究顯示，DUBs在動(dòng)物生殖細(xì)胞減數(shù)分裂過(guò)程中發(fā)揮關(guān)鍵作用。例如，DUBs中的泛素羧基末端水解酶家族(ubiquitin C-terminal hydrolases，UCHs)通過(guò)調(diào)節(jié)卵母細(xì)胞發(fā)育及紡錘體形成，于哺乳動(dòng)物卵母細(xì)胞成熟至關(guān)重要。Uchl-1和Uchl-3 mRNA在小鼠及恒河猴卵母細(xì)胞GV期和M Ⅱ中期高表達(dá)，其中，Uchl-1與卵母細(xì)胞皮質(zhì)有關(guān)，Uchl-3則與減數(shù)分裂紡錘體形成相關(guān)。GV期卵母細(xì)胞微注射UCHs抑制劑——泛素醛(ubiquitin-aldehyde，UBAL)導(dǎo)致大部分細(xì)胞不能進(jìn)入M Ⅰ中期，并出現(xiàn)紡錘體和第一極體排出的異常，注射Uchl-3抗體影響卵母細(xì)胞成熟并導(dǎo)致紡錘體形態(tài)異常及減數(shù)分裂異常[63]。此外，UCHs缺乏卵母細(xì)胞受精率降低，且突變體胚胎無(wú)法形成囊胚[64]。UCHs在精子形成中也發(fā)揮重要生理作用[65]，例如，Uchl-1在精原干細(xì)胞有絲分裂增殖中發(fā)揮作用，Uchl-3則在精母細(xì)胞減數(shù)分裂及附睪精子成熟發(fā)揮作用[65]。研究顯示Uchl-1對(duì)精子發(fā)生過(guò)程中細(xì)胞凋亡的調(diào)節(jié)具有重要作用，小鼠睪丸過(guò)表達(dá)Uchl-1導(dǎo)致精母細(xì)胞凋亡數(shù)量增加，出現(xiàn)精母細(xì)胞粗線(xiàn)期阻滯[66]，敲除Uchl-1導(dǎo)致PND7-14減數(shù)分裂前生殖細(xì)胞數(shù)量及凋亡蛋白TRP53、Bax及caspase-3等的水平增加[67]。

6 小 結(jié)

UPP在生殖細(xì)胞減數(shù)分裂過(guò)程不可或缺，為保障生殖細(xì)胞基因組完整性及雌雄配子正常發(fā)育的遺傳基礎(chǔ)，其相關(guān)組件缺失及突變導(dǎo)致減數(shù)分裂重組、MSCI及細(xì)胞周期進(jìn)程阻滯等缺陷。此外，UPP在精子頂體形成、精子尾部發(fā)育、精卵結(jié)合、合子中父系線(xiàn)粒體降解及母系蛋白降解等過(guò)程發(fā)揮重要生理調(diào)控作用。研究顯示，眾多UPP組件在生殖細(xì)胞不同發(fā)育階段發(fā)揮作用，同一UPP組件在雌性生殖細(xì)胞和雄性生殖細(xì)胞發(fā)育中具有不同的調(diào)控作用，同一E2與不同的E3結(jié)合也發(fā)揮不同生理作用，UPP這一復(fù)雜而精確的調(diào)控機(jī)制是保障生殖細(xì)胞正常減數(shù)分裂過(guò)程并產(chǎn)生具有受精/授精能力的配子的基礎(chǔ)。目前，針對(duì)UPP對(duì)生殖細(xì)胞減數(shù)分裂影響的研究主要集中在小鼠和人類(lèi)中，而在豬生殖過(guò)程中的研究相對(duì)較少。因此，對(duì)UPP在豬生殖細(xì)胞減數(shù)分裂過(guò)程中的生化特性、亞細(xì)胞定位、相關(guān)底物及其在豬配子生成過(guò)程中發(fā)揮的生物學(xué)功能和具體作用機(jī)制的研究，能進(jìn)一步推進(jìn)對(duì)其生殖功能的了解，對(duì)提高其繁殖性能具有十分重要的意義。

[1] LI C H,XIAO Z X.Regulation of p63 protein stability via ubiquitin-proteasome pathway[J].BioMed Research International,2014,2014:175721.

[2] HAO Y H,DOYLE J M,RAMANATHAN S,et al.Regulation of WASH-dependent actin polymerization and protein trafficking by ubiquitination[J].Cell,2013,152(5):1051-1064.

[3] PETERS J M,HARRIS R,FINLEY D.Ubiquitin and the biology of the cell[M].New York:Springer Science & Business Media,2013:230.

[4] TU Y Q,CHEN C,PAN J R,et al.The ubiquitin proteasome pathway (UPP) in the regulation of cell cycle control and DNA damage repair and its implication in tumorigenesis[J].International Journal of Clinical & Experimental Pathology,2012,5(8):726-738.

[5] YOSHIZAWA T,KARIM M F,SATO Y,et al.SIRT7 controls hepatic lipid metabolism by regulating the ubiquitin-proteasome pathway[J].Cell Metabolism,2014,19(4):712-721.

[6] BHAT M,KALAM R,QADRI S S Y H,et al.Vitamin D deficiency-induced muscle wasting occurs through the ubiquitin proteasome pathway and is partially corrected by calcium in male rats[J].Endocrinology,2013,154(11):4018-4029.

[7] HAMILTON A M,ZITO K.Breaking it down:the ubiquitin proteasome system in neuronal morphogenesis[J].Neural Plasticity,2013,3:196848.

[8] AL-QUSAIRI L,PROKIC I,AMOASII L,et al.Lack of myotubularin (MTM1) leads to muscle hypotrophy through unbalanced regulation of the autophagy and ubiquitin-proteasome pathways[J].The FASEB Journal,2013,27(8):3384-3394.

[9] ZHANG L P,TANG H,KOU Y,et al.MG132-mediated inhibition of the ubiquitin-proteasome pathway ameliorates cancer cachexia[J].Journal of Cancer Research and Clinical Oncology,2013,139(7):1105-1115.

[10] CHEN F Z,ZHAO X K.Ubiquitin-proteasome pathway and prostate cancer[J].Oncology Research and Treatment,2013,36(10):592-596.

[11] VYAS R,KUMAR R,CLERMONT F,et al.RNF4 is required for DNA double-strand break repairinvivo[J].Cell Death and Differentiation,2013,20(3):490-502.

[12] AN J Y,KIM E A,JIANG Y,et al.UBR2 mediates transcriptional silencing during spermatogenesis via histone ubiquitination[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(5):1912-1917.

[13] OH J S,HAN S J,CONTI M.Wee1B,Myt1,and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption[J].The Journal of Cell Biology,2010,188(2):199-207.

[14] POMERANTZ Y,DEKEL N.Molecular participants in regulation of the meiotic cell cycle in mammalian oocytes[J].Reproduction,Fertility and Development,2013,25(3):484-494.

[15] SHIN S W,SHIMIZU N,TOKORO M,et al.Mouse zygote-specific proteasome assembly chaperone important for maternal-to-zygotic transition[J].Biology Open,2012,2(2):170-182.

[16] KOPANJA D,ROY N,STOYANOVA T,et al.Cul4A is essential for spermatogenesis and male fertility[J].Developmental Biology,2011,352(2):278-287.

[17] WYKES S M,KRAWETZ S A.The structural organization of sperm chromatin[J].Journal of Biological Chemistry,2003,278(32):29471-29477.

[18] YU C,JI S Y,SHA Q Q,et al.CRL4-DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation[J].Nature Communications,2015,6:8017.

[19] KIRENBERG I,SHAHAR-POMERANTZ Y,ELBAZ J,et al.New insights into the ubiquitin-proteasome pathway in oocytes resuming meiosis[J].Biology of Reproduction,2012,87(Suppl.1):126.

[20] KLEIGER G,MAYOR T.Perilous journey:a tour of the ubiquitin-proteasome system[J].Trends in Cell Biology,2014,24(6):352-359.

[21] IKEDA F,DIKIC I.Atypical ubiquitin chains:new molecular signals.Protein modifications[J].Croatian Medical Journal,2008,9(6):536-542.

[22] NIR I,HUTTNER D,MELLER A.Direct sensing and discrimination among ubiquitin and ubiquitin chains using solid-state nanopores[J].Biophysical Journal,2015,108(9):2340-2349.

[23] 沈夷清.細(xì)胞周期調(diào)控中APC/C降解機(jī)制的研究[D].碩士學(xué)位論文.天津:河北工業(yè)大學(xué),2014:57-58.

[24] JESSUS C.MPF and the control of meiotic divisions:old problems,new concepts[J].Oogenesis:The Universal Process,2010:227-265.

[25] MARANGOS P,CARROLL J.Securin regulates entry into M-phase by modulating the stability of cyclin B[J].Nature Cell Biology,2008,10(4):445-451.

[26] MAGANGOS P,VERSCHUREN E W,CHENH R,et al.Prophase Ⅰ arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APCCdh1[J].The Journal of Cell Biology,2007,176(1):65-75.

[27] BEN-ELIEZER I,POMERANTZ Y,GALIANI D,et al.Appropriate expression of Ube2C and Ube2S controls the progression of the first meiotic division[J].The FASEB Journal,2015,29(11):4670-4681.

[28] 劉相元,胡弘歷,歐陽(yáng)華芳,等.CRL E3泛素連接酶復(fù)合體研究進(jìn)展[J].中國(guó)細(xì)胞生物學(xué)學(xué)報(bào),2014,36(2):157-168.

[29] YIN Y,LIN C X,KIM S T,et al.The E3 ubiquitin ligase Cullin 4A regulates meiotic progression in mouse spermatogenesis[J].Developmental Biology,2011,356(1):51-62.

[30] REIS A,MADGWICK S,CHANG H Y,et al.Prometaphase APCcdh1 activity prevents non-disjunction in mammalian oocytes[J].Nature Cell Biology,2007,9(10):1192-1198.

[31] DEANTONI A,SALA V,MUSACCHIO A.Explaining the oligomerization properties of the spindle assembly checkpoint protein Mad2[J].Philosophical Transactions of the Royal Society of London B:Biological Sciences,2005,360(1455):637-648.

[32] ZHOU L,YANG Y,ZHANG J J,et al.The role of RING box protein 1 in mouse oocyte meiotic maturation[J].PLoS One,2013,8(7):e68964.

[33] JOSEFSBERG L B Y,GALIANI D,DANTES A,et al.The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes[J].Biology of Reproduction,2000,62(5):1270-1277.

[34] CLOUTIER J M,TURNER J M A.Meiotic sex chromosome inactivation[J].Current Biology,2010,20(22):R962-R963.

[35] TURNER J M A.Meiotic sex chromosome inactivation[J].Development,2007,134(10):1823-1831.

[36] BAARENDS W M,HOOGERBRUGGE J W,ROEST H P,et al.Histone ubiquitination and chromatin remodeling in mouse spermatogenesis[J].Developmental Biology,1999,207(2):322-333.

[37] WANG H,WANG L J,ERDJUMENT-BROMAGE H,et al.Role of histone H2A ubiquitination in polycomb silencing[J].Nature,2004,431(7010):873-878.

[38] AN J Y,KIM E,ZAKRZEWSKA A,et al.UBR2 of the N-end rule pathway is required for chromosome stability via histone ubiquitylation in spermatocytes and somatic cells[J].PLoS One,2012,7(5):e37414.

[39] VASKOVA E A,PAVLOVA S V,SHEVCHENKO A I,et al.Meiotic inactivation of sex chromosomes in mammals[J].Russian Journal of Genetics,2010,46(4):385-393.

[40] INAGAKI A,SCHOENMAKERS S,BAARENDS W M.DNA double strand break repair,chromosome synapsis and transcriptional silencing in meiosis[J].Epigenetics,2010,5(4):255-266.

[41] INAGAKI A,SLEDDENS-LINKELS E,WASSENAAR E,et al.Meiotic functions of RAD18[J].Journal of Cell Science,2011,124(16):2837-2850.

[42] MA T,KELLER J A,YU X C.RNF8-dependent histone ubiquitination during DNA damage response and spermatogenesis[J].Acta Biochimica et Biophysica Sinica,2011,43(5):339-345.

[43] GILLOT I,MATTHEWS C,PUEL D,et al.Ret finger protein:an E3 ubiquitin ligase juxtaposed to the XY body in meiosis[J].International Journal of Cell Biology,2009,2009:524858.

[44] HOGARTH C A,MITCHELL D,EVANOFF R,et al.Identification and expression of potential regulators of the mammalian mitotic-to-meiotic transition[J].Biology of Reproduction,2011,84(1):34-42.

[45] GUARDAVACCARO D,KUDO Y,BOULAIRE J,et al.Control of meiotic and mitotic progression by the F box protein β-Trcp1invivo[J].Developmental Cell,2003,4(6):799-812.

[46] LU C,KIM J,FULLER M T.The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila[J].Development,2013,140(17):3522-3531.

[47] WANG Y L,LI D,YANG H D,et al.The E3 ubiquitin ligase CRL4 regulates proliferation and progression through meiosis in Chinese mitten crabEriocheirsinensis[J].Biology of Reproduction,2001,94(3):65.

[48] TURCO E,GALLEGO L D,SCHNEIDER M,et al.Monoubiquitination of histone H2B is intrinsic to the Bre1 RING domain-Rad6 interaction and augmented by a second Rad6-binding site on Bre1[J].Journal of Biological Chemistry,2015,290(9):5298-5310.

[49] GAME J C,CHERNIKOVA S B.The role of RAD6 in recombinational repair,checkpoints and meiosis via histone modification[J].DNA Repair,2009,8(4):470-482.

[50] YAMASHITA K,SHINOHARA M,SHINOHARA A.Rad6-Bre1-mediated histone H2B ubiquitylation modulates the formation of double-strand breaks during meiosis[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(31):11380-11385.

[51] KIM J,GUERMAH M,MCGINTY R K,et al.RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells[J].Cell,2009,137(3):459-471.

[52] BAARENDS W M,WASSENAAR E,HOOGERBRUGGE J W,et al.Increased phosphorylation and dimethylation of XY body histones in the Hr6b-knockout mouse is associated with derepression of the X chromosome[J].Journal of Cell Science,2007,120(11):1841-1851.

[53] BAARENDS W M,WASSENAAR E,HOOGERBRUGGE J W,et al.Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase[J].Molecular and Cellular Biology,2003,23(4):1151-1162.

[54] TASAKI T,KWON Y T.The mammalian N-end rule pathway:new insights into its components and physiological roles[J].Trends in Biochemical Sciences,2007,32(11):520-528.

[55] KWON Y T,XIA Z X,AN J Y,et al.Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-end rule pathway[J].Molecular and Cellular Biology,2003,23(22):8255-8271.

[56] MIYAMOTO T,TSUJIMURA A,MIYAGAWA Y,et al.Single nucleotide polymorphism in theUBR2 gene may be a genetic risk factor for Japanese patients with azoospermia by meiotic arrest[J].Journal of Assisted Reproduction and Genetics,2011,28(8):743-746.

[57] CUI L,LI W.Role of ubiquitination in meiotic recombination repair[J].Science China Life Sciences,2010,53(4):447-454.

[58] PINATO S,SCANDIUZZI C,ARNAUDO N,et al.RNF168,a new RING finger,MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX[J].BMC Molecular Biology,2009,10(1):55.

[59] PANIER S,DUROCHER D.Regulatory ubiquitylation in response to DNA double-strand breaks[J].DNA Repair,2009,8(4):436-443.

[60] POLANOWSKA J,MARTIN J S,GARCIA-MUSE T,et al.A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites[J].The EMBO Journal,2006,25(10):2178-2188.

[61] POULSEN M,LUKAS C,LUKAS J,et al.Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks[J].The Journal of Cell Biology,2012,197(2):189-199.

[62] POMERANTZ Y,ELBAZ J,BEN-ELIEZER I,et al.From ubiquitin-proteasomal degradation to CDK1 inactivation:requirements for the first polar body extrusion in mouse oocytes[J].The FASEB Journal,2012,26(11):4495-4505.

[63] MTANGO N R,SUTOVSKY M,VANDEVOORT C A,et al.Essential role of ubiquitin C-terminal hydrolases UCHL1 and UCHL3 in mammalian oocyte maturation[J].Journal of Cellular Physiology,2012,227(5):2022-2029.

[64] MTANGO N R,LATHAM K E,SUTOVSKY P.Deubiquitinating enzymes in oocyte maturation,fertilization and preimplantation embryo development[M]//SUTOVSKY P.Posttranslational protein modifications in the reproductive system.New York:Springer,2014:45-48.

[65] KWON J,WANG Y L,SETSUIE R,et al.Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice[J].Biology of Reproduction,2004,71(2):515-521.

[66] WANG Y L,LIU W Z,SUN Y J,et al.Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice[J].Molecular Reproduction and Development,2006,73(1):40-49.

[67] KWON J,MOCHIDA K,WANG Y L,et al.Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis[J].Biology of Reproduction,2005,73(1):29-35.

*Corresponding author, professor, E-mail: chenbin7586@126.com