陳會(huì)友，張建敏，李柏森，鄧永琳，張龔煒

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犏牛雄性不育的減數(shù)分裂基因表達(dá)與表觀遺傳調(diào)控研究進(jìn)展

陳會(huì)友，張建敏，李柏森，鄧永琳，張龔煒

西南大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，重慶 402460

種間雜交雄性不育是自然界普遍現(xiàn)象，是物種形成生殖隔離的重要方式。犏牛作為牦牛()和普通牛()的種間雜交后代，表現(xiàn)為公犏牛不育，而母犏?？捎?，是研究種間雜交雄性不育的良好動(dòng)物模型。近年來利用分子生物學(xué)技術(shù)發(fā)現(xiàn)犏牛睪丸組織中大量基因表達(dá)紊亂。研究表明，DNA甲基化、組蛋白修飾和非編碼RNA等表觀遺傳因素參與精子發(fā)生過程。本文從減數(shù)分裂相關(guān)的基因表達(dá)、DNA甲基化、microRNA (miRNA)、PIWI蛋白相互作用的RNA (PIWI-interactingRNA, piRNA)、長鏈非編碼RNA (long non-coding RNA, lncRNA)和組蛋白甲基化修飾等方面總結(jié)了犏牛雄性不育的相關(guān)研究進(jìn)展，以期從遺傳和表觀遺傳調(diào)控角度更加深入理解犏牛雄性不育的分子機(jī)理。

犏牛；雄性不育；表觀遺傳；基因表達(dá)

牦牛()被稱為“高原之舟”，是青藏高原地區(qū)不可或缺的全能家畜，對(duì)高寒、缺氧和強(qiáng)紫外線等惡劣的生態(tài)環(huán)境條件有極強(qiáng)的適應(yīng)性，是當(dāng)?shù)鼐用癫豢苫蛉钡纳a(chǎn)資料和生活資料。但是牦牛乳、肉用生產(chǎn)性能較低。為了改善牦牛生產(chǎn)性能，利用牦牛和優(yōu)良肉用、奶用普通牛()開展種間雜交，F(xiàn)1、F2代犏牛具有明顯雜種優(yōu)勢(shì)，肉用、奶用均比親本牦牛有顯著提高并能適應(yīng)高原地區(qū)環(huán)境氣候。但是，F(xiàn)1、F2代犏牛雄性不育，這使得其雜種優(yōu)勢(shì)無法通過橫交固定，無法通過雜交育種改良牦牛品種，成為阻礙藏區(qū)牦牛產(chǎn)業(yè)發(fā)展的瓶頸問題。種間雜交雄性不育是自然界普遍現(xiàn)象，是物種形成生殖隔離的重要方式。犏牛雄性不育也是探索物種形成生殖隔離的良好動(dòng)物模型。目前，國內(nèi)外學(xué)者已從雜交改良、組織學(xué)、內(nèi)分泌學(xué)、生物化學(xué)、細(xì)胞遺傳學(xué)和分子生物學(xué)等主要領(lǐng)域展開研究[1,2]。最近研究表明，DNA甲基化、組蛋白修飾和非編碼RNA等表觀遺傳因素是調(diào)控基因表達(dá)的重要因子，并對(duì)精子發(fā)生過程起關(guān)鍵作用[3]。本文從基因表達(dá)、DNA甲基化、組蛋白甲基化修飾和非編碼RNA等方面總結(jié)了犏牛雄性不育的相關(guān)研究進(jìn)展，以期從遺傳和表觀遺傳調(diào)控角度更加深入理解犏牛雄性不育的分子機(jī)理。

1 基因表達(dá)紊亂與犏牛雄性不育

組織學(xué)研究發(fā)現(xiàn)，犏牛雄性不育主要表現(xiàn)為精母細(xì)胞數(shù)量減少，生精小管內(nèi)極少見精子細(xì)胞，表明犏牛雄性不育主要是生精細(xì)胞減數(shù)分裂過程受阻[4]。減數(shù)分裂是有性生殖過程中產(chǎn)生配子的一種特殊分裂方式，是哺乳動(dòng)物繁衍后代的必須條件。研究人員在減數(shù)分裂相關(guān)基因上展開大量研究，以探索減數(shù)分裂基因表達(dá)紊亂與犏牛雄性不育的關(guān)系，其研究主要集中在以下蛋白家族(表1)。

表1 犏牛雄性不育相關(guān)基因

1.1 DAZ (deleted in azoospermia)蛋白家族

DAZ蛋白家族主要有3個(gè)成員，(the deleted in azoospermia)基因位于Y染色體上，(deleted in azoospermia like)和(boule protein)是常染色體基因，這3個(gè)基因編碼蛋白是RNA結(jié)合蛋白，在生殖細(xì)胞特異表達(dá)，是精子發(fā)生過程的主要調(diào)控因子。有4個(gè)拷貝，以頭對(duì)頭的形式排列在Y染色體上，基因產(chǎn)物具有RNA結(jié)合蛋白特性，在睪丸組織特異性表達(dá)，可能與精子生成有關(guān)，是決定精子生成的基因[5]。與基因同源性約為83％，在黃牛()和牦牛睪丸組織中表達(dá)，在F1代犏牛睪丸組織不表達(dá)，并且的DNA甲基化程度顯著高于黃牛和牦牛[6]。這與小鼠()基因敲除后導(dǎo)致精子發(fā)生停止或功能異常結(jié)果一致[7]，說明基因可能對(duì)犏牛雄性不育有重要影響。BOULE是動(dòng)物精母細(xì)胞減數(shù)分裂過程中的必需蛋白(圖1)，與精子發(fā)生減數(shù)分裂阻滯、雄性不育等密切相關(guān)，F(xiàn)1代犏牛基因表達(dá)水平顯著低于黃牛，5?端DNA甲基化水平極顯著高于黃牛和牦牛[8]，說明犏?；虻母呒谆赡苁蛊鋗RNA表達(dá)下調(diào)，對(duì)犏牛生精細(xì)胞減數(shù)分裂、雄性不育有重要影響。

1.2 SYCP (synaptonemal complex protein)蛋白家族

SYCP蛋白家族主要有4個(gè)成員，分別是SYCP1 (synaptonemal complex protein 1)、SYCP2、SYCP3和FKBP6 (FKBP prolyl isomerase 6)。它們參與精原細(xì)胞的形成，是聯(lián)會(huì)復(fù)合體形成的關(guān)鍵蛋白。主要在減數(shù)分裂前期表達(dá)，在同源染色體配對(duì)中發(fā)揮重要作用，是精子發(fā)生過程中所必須的基因。在粗線期和雙線期，F(xiàn)KBP6與SYCP1蛋白共同結(jié)合于常染色體的聯(lián)會(huì)區(qū)域，輔助SYCP1形成橫絲(transverse filaments, TF)。在犏牛、牦牛和普通牛中都有表達(dá)，且差異不顯著[9]。SYCP2蛋白能與SYCP3蛋白結(jié)合發(fā)揮作用。在哺乳動(dòng)物中，SYCP2和SYCP3是軸向元件(axial element, AE)和側(cè)向元件(lateral elements, LE)形成的主要決定成分[10]。SYCP3蛋白是一個(gè)DNA結(jié)合蛋白，定位于聯(lián)會(huì)復(fù)合體的側(cè)成分，在睪丸中特別是在初級(jí)精母細(xì)胞中表達(dá)(圖1)，在同源染色體配對(duì)中發(fā)揮重要作用，是精子發(fā)生過程中減數(shù)分裂所必須。犏牛睪丸表達(dá)顯著低于牦牛[11]，其DNA啟動(dòng)子甲基化水平顯著高于牦牛[12]。主要在性腺組織的粗線期表達(dá)，犏牛睪丸表達(dá)量顯著低于牦牛。因此，、和基因表達(dá)紊亂與犏牛雄性不育存在一定聯(lián)系[13]。

圖1 犏牛睪丸中的生精調(diào)控

1.3 DEAD-box (DEAD-box helicase)蛋白家族

DEAD-box蛋白家族是一個(gè)ATP依賴的RNA解旋酶家族，參與多種RNA代謝過程。其中(DEAD-box helicase 4)、(DEAD-box helicase 3, Y-linked)和是與精子發(fā)生密切相關(guān)的基因。在哺乳動(dòng)物中，、和的缺失或減少會(huì)導(dǎo)致不同形式的精子發(fā)生障礙。DDX4在哺乳動(dòng)物生殖系特異表達(dá)，作為一種廣泛的分子標(biāo)記物被應(yīng)用于生殖研究[14]。犏牛睪丸組織的啟動(dòng)子區(qū)甲基化水平顯著高于牦牛，其mRNA在犏牛睪丸中的表達(dá)量也極顯著低于牦牛[15]，說明對(duì)犏牛雄性不育存在一定影響。位于牛Y染色體上，在3種牛睪丸組織中mRNA表達(dá)量差異不顯著[16]。DDX25是已知的唯一由激素調(diào)節(jié)的RNA解旋酶，基因敲除小鼠精子發(fā)生受阻，致使圓形精子無法繼續(xù)變形[17]。犏牛睪丸組織中的基因表達(dá)水平也顯著低于牦牛[18]，可能致使犏牛精子發(fā)生受阻。

1.4 減數(shù)分裂同源重組相關(guān)基因

在染色體自我復(fù)制過程中可能會(huì)出現(xiàn)雙鏈斷裂(double strand break, DSB)現(xiàn)象，如果這些斷裂未能及時(shí)修復(fù)就會(huì)引起細(xì)胞凋亡，修復(fù)不準(zhǔn)確也會(huì)引起基因突變和染色體突變。真核生物對(duì)這些斷裂的修復(fù)有兩種機(jī)理：非同源末端連接和同源重組，而同源重組是DNA上DSB損傷修復(fù)的主要方式，對(duì)于保持哺乳動(dòng)物細(xì)胞的基因組完整性十分重要[19]。(DNA meiotic recombinase 1)、(replication protein A1)和(BLM RecQ like helicase)等是參與哺乳動(dòng)物減數(shù)分裂同源重組修復(fù)的關(guān)鍵基因。這些基因的突變、敲除和表達(dá)水平的降低均會(huì)引起精母細(xì)胞減數(shù)分裂障礙，最終導(dǎo)致雄性不育。、基因在犏牛睪丸組織中的表達(dá)水平對(duì)比黃牛和牦牛差異顯著[20~22]，且基因DNA啟動(dòng)子區(qū)甲基化水平也差異顯著，提示其可能和犏牛雄性不育相關(guān)。

1.5 Y染色體雄性特異區(qū)(male-specific region on the Y, MSY)相關(guān)基因

Y染色體是雄性哺乳動(dòng)物相對(duì)于雌性特有的染色體，由常染色體進(jìn)化而來，和X染色體只有5%的區(qū)域相同，該區(qū)域是和X染色體同源重組的擬常染色體區(qū)，而95%的其他區(qū)域則是MSY (圖1)。牛Y染色體基因組已經(jīng)公布(NCBI GenBank accession no. CM001061)，通過對(duì)牛Y染色體進(jìn)行測序和注釋，牛Y染色體共鑒定出1274個(gè)基因，MSY包含28個(gè)蛋白編碼基因和375個(gè)新轉(zhuǎn)錄本。利用轉(zhuǎn)錄組測序(RNA-seq)技術(shù)比較普通牛不同年齡睪丸組織中基因表達(dá)模式發(fā)現(xiàn)，13個(gè)編碼基因和220轉(zhuǎn)錄本的表達(dá)量隨睪丸發(fā)育顯著上調(diào)，這表明Y染色體MSY區(qū)域的基因參與牛生精過程[23]。

MSY相關(guān)基因的拷貝數(shù)變異(copy number variation, CNV)被證明和雄性生精功能有關(guān)[24]。張龔煒等[25]首次對(duì)MSY相關(guān)基因的CNV進(jìn)行研究，發(fā)現(xiàn)F1、F2代公犏牛MSY相關(guān)基因(testis specific protein, Y-linked)、(heat shock transcription factor, Y-linked)、(preferentially expressed antigen in melanoma, Y-linked)和(zinc finger protein 280B, Y-linked)的幾何平均拷貝數(shù)(the average geometric mean copy number, CN)顯著高于普通牛和牦牛，提示犏牛MSY在基因組結(jié)構(gòu)上和牦牛以及普通牛不同，MSY相關(guān)基因的CNV可能是犏牛雄性不育的原因。隨后詳細(xì)分析普通牛和牦牛MSY相關(guān)基因、、、和序列，發(fā)現(xiàn)只有在?？剖潜Ｊ氐?，牦牛缺失類型序列，和在牦牛Y染色體上成功擴(kuò)增，和的平均拷貝數(shù)在普通牛和牦牛之間差異顯著[4]，說明普通牛和牦牛MSY存在差異性。犏牛、、和表達(dá)對(duì)比牦牛和普通牛顯著下調(diào)，究其原因可能是犏牛精子發(fā)生異常導(dǎo)致無精子生成，提示這些基因主要參與減數(shù)分裂后精子形成過程[23,4]。除以上多拷貝基因外，犏牛睪丸組織MSY區(qū)域的單拷貝基因(ubiquitously transcribed tetratricopeptide repeat con-taining, Y-linked)、(oral-facial-digital syndrome 1, Y-linked)和(ubiquitin specific peptidase 9, Y-linked)表達(dá)對(duì)比牦牛和普通牛顯著上調(diào)。和具有組蛋白甲基化和泛素化的功能，提示后續(xù)可進(jìn)一步從組蛋白甲基化和泛素化角度探索犏牛雄性不育的機(jī)理[16]。

2 表觀遺傳與犏牛雄性不育

以DNA甲基化、組蛋白修飾和染色質(zhì)重塑為特征的表觀遺傳修飾是包括精子發(fā)生在內(nèi)的許多生物學(xué)過程中的重要調(diào)節(jié)因子[3]。DNA甲基化[41]、組蛋白修飾[42]和非編碼RNA[43]作為機(jī)體重要的表觀遺傳修飾類型，是在精子發(fā)生過程中調(diào)控的關(guān)鍵因素。表觀遺傳修飾的異常，將使生精過程基因的表達(dá)紊亂，進(jìn)而導(dǎo)致雄性不育。

2.1 DNA甲基化與犏牛雄性不育

DNA甲基化是在甲基轉(zhuǎn)移酶(DNA methyltrans-ferase, DNMT)催化作用下，以S-腺苷甲硫氨酸(S-Adenosylmethionine, SAM)作為甲基供體，通過共價(jià)鍵結(jié)合的方式使基因組CpG二核苷酸中胞嘧啶5號(hào)位碳原子獲得一個(gè)甲基基團(tuán)的化學(xué)修飾過程[44]。DNA甲基化能引起染色質(zhì)結(jié)構(gòu)、DNA構(gòu)象、DNA穩(wěn)定性及DNA與蛋白質(zhì)相互作用方式的改變，從而調(diào)控基因表達(dá)[45]。DNA甲基化可能通過調(diào)節(jié)雄性生殖細(xì)胞的增殖和分化而發(fā)揮關(guān)鍵作用[46]，是雄性不育的一個(gè)重要影響因素。在雄性不育模型中觀察到的異常DNA甲基化模式可能是精原細(xì)胞的再甲基化失敗或精母細(xì)胞、精子細(xì)胞和成熟精子細(xì)胞的甲基化狀態(tài)維持不變所致[47]。由于啟動(dòng)子DNA甲基化通常抑制基因轉(zhuǎn)錄，在精子發(fā)生過程中DNA甲基化的紊亂導(dǎo)致了生精基因的表達(dá)紊亂，和雄性不育高度相關(guān)[45]。在犏牛中，研究發(fā)現(xiàn)、和基因啟動(dòng)子區(qū)域甲基化水平升高導(dǎo)致基因表達(dá)下調(diào)，進(jìn)而影響犏牛生殖[48~50]。最近利用全基因組甲基化測序技術(shù)發(fā)現(xiàn)啟動(dòng)子高甲基化基因在配子產(chǎn)生、piRNA (非編碼RNA的一種)代謝過程和染色質(zhì)結(jié)構(gòu)的DNA甲基化過程中顯著富集，表明啟動(dòng)子高甲基化和piRNA途徑等表觀遺傳紊亂可能和犏牛雄性不育高度相關(guān)[29]。犏牛睪丸中PIWI/piRNA通路基因啟動(dòng)子發(fā)生DNA超甲基化，使、、(phospholipase D family member 6)、(maelstrom spermatogenic transposon silencer)、、(tudor domain containing 1)和等基因表達(dá)下調(diào)，并導(dǎo)致犏牛精子發(fā)生過程中粗線期piRNA的產(chǎn)生降低，同時(shí)還發(fā)現(xiàn)轉(zhuǎn)座因子LINEs (long interspersed nuclear elements)、SINEs (short interspersed nuclear elements)和LTRs (long terminal repeats)在犏牛睪丸中高甲基化(圖1)。因此，DNA高甲基化和piRNA生成途徑中斷是導(dǎo)致生殖細(xì)胞發(fā)育不成功的原因之一，這可能導(dǎo)致犏牛雄性不育[29]。

2.2 非編碼RNA與犏牛雄性不育

隨著基因組研究的深入，以前普遍認(rèn)為不編碼蛋白質(zhì)的非編碼RNA被證明其在轉(zhuǎn)錄后具有基因調(diào)控的作用。非編碼RNA主要包括lncRNA、核糖體RNA (rRNA)、轉(zhuǎn)運(yùn)RNA (tRNA)、核小RNA (small nuclearRNA, snRNA)、核仁小RNA (small nucleolar RNA, snoRNA)、miRNA和piRNA等多種已知功能的RNA，及未知功能的RNA。這些RNA的共同特點(diǎn)是都能從基因組上轉(zhuǎn)錄而來，但是不翻譯成蛋白，在RNA水平上就能行使各自的生物學(xué)功能。例如piRNA主要在睪丸組織中表達(dá)，其在轉(zhuǎn)錄后水平調(diào)控動(dòng)物生殖系統(tǒng)[51~53]。

2.2.1 PIWI/piRNA途徑與犏牛雄性不育

piRNA一般長約24~35 nt，通過與AGO蛋白家族相互作用形成piRNA沉默復(fù)合體(piRNA-induced silencing complex, pi-RISC)來調(diào)控基因重復(fù)序列及轉(zhuǎn)座子等基因元件的活性[54]，主要影響動(dòng)物生殖[51]。小鼠減數(shù)分裂時(shí)期piRNAs主要分前粗線期piRNAs (26~28 nt)和粗線期piRNAs (~30 nt)[55]。前粗線期piRNAs主要在細(xì)線期精母細(xì)胞中發(fā)現(xiàn)，來源于轉(zhuǎn)座子元件[56,57]。粗線期piRNAs起源于基因組不同區(qū)域的piRNA簇，并與粗線期精母細(xì)胞中的PIWIL1和PIWIL2結(jié)合維持到圓形精子細(xì)胞階段，是成年小鼠睪丸中的主要piRNAs，約占總piRNAs的95%[58]。在小鼠中，編碼PIWI蛋白的/、和/基因以及其他協(xié)助piRNA產(chǎn)生或功能表達(dá)所必需的蛋白是PIWI/piRAN通路的重要組成部分，它們共同維持小鼠的精子發(fā)生。一旦piRNA通路基因發(fā)生突變，都會(huì)導(dǎo)致雄性不育[59,60]。例如，轉(zhuǎn)錄因子A-MYB/MYBL1 (myelo-blastosis oncogene-like 1)被證明直接識(shí)別上游DNA元件來啟動(dòng)粗線期piRNAs的轉(zhuǎn)錄[55]，生成的piRNA前體轉(zhuǎn)錄本被輸送到核外細(xì)胞質(zhì)，隨后又被PIWI蛋白結(jié)構(gòu)域修剪成前體piRNA，經(jīng)過一系列修剪后，由核酸外切酶(PARN like, ribonuclease domain con-taining 1, PNLDC1)[61]對(duì)piRNA3?端進(jìn)一步修剪成成熟大小，最終piRNA3?端被2?-O-甲基轉(zhuǎn)移酶(HEN methyltransferase 1, HENMT1)修飾產(chǎn)生成熟的piRNA[62](圖2)。如前所述，犏牛睪丸中PIWI/piRNA通路相關(guān)基因(、、、、、和)啟動(dòng)子超甲基化抑制基因表達(dá)，使piRNA生成顯著降低[29]，表明PIWI/piRNA通路參與犏牛雄性不育的過程。

2.2.2 miRNA與犏牛雄性不育

miRNA是一種長度約為22 nt并高度保守的內(nèi)源性非編碼小分子RNA，雖然不編碼蛋白質(zhì)但具有調(diào)控功能。miRNA的生物學(xué)功能主要體現(xiàn)在對(duì)其靶基因的轉(zhuǎn)錄后水平調(diào)控，主要有靶標(biāo)mRNA的降解和mRNA翻譯抑制2種方式，它們?cè)诰影l(fā)生過程中以相對(duì)特異性方式表達(dá)，在雄性動(dòng)物生殖健康中起著至關(guān)重要的作用[63]。例如，miRNA參與調(diào)控睪丸支持細(xì)胞的增殖和粘附，一旦支持細(xì)胞增殖和粘附功能異常，將會(huì)導(dǎo)致精子發(fā)生受阻[64]。因此，miRNAs的失調(diào)被認(rèn)為是雄性不育的分子基礎(chǔ)，這些分子的異常表達(dá)模式可以遺傳給后代[65]。研究表明，miRNA在哺乳動(dòng)物精子發(fā)生的不同過程中起著重要的調(diào)節(jié)作用：和通過靶向(signal transducer and activator of transcription 3)和(Cyclin D1)在轉(zhuǎn)錄后水平促進(jìn)小鼠 SSCs的更新[66]，通過抑制(KIT proto-oncogene, receptor tyrosine kinase)的表達(dá)在維持精原細(xì)胞的未分化狀態(tài)中發(fā)揮關(guān)鍵作用[67]。研究表明，小鼠粗線期精母細(xì)胞中存在許多來自X染色體的miRNAs，這些miRNAs可能導(dǎo)致性染色體減數(shù)分裂失活[68]。徐傳飛[69]通過對(duì)小RNA測序發(fā)現(xiàn)61個(gè)miRNA在犏牛與牦牛睪丸組織之間差異表達(dá)，其中、、、和參與SSCs自我更新以及分化過程；和參與精子發(fā)生過程中減數(shù)分離起始過程，說明miRNA在犏牛生精過程中具有影響力。廖珂[70]發(fā)現(xiàn)涉及細(xì)胞增殖、凋亡過程的miRNA表達(dá)在犏牛和普通牛間也存在差異，如、和等。徐傳飛[71]對(duì)差異miRNAs的靶基因進(jìn)行GO分析和KEGG分析，結(jié)果顯示靶向的(cyclin dependent kinase 2)、和主要參與細(xì)胞分化、增殖以及凋亡等通路。

圖2 粗線期piRNA的生物發(fā)生

2.2.3 lncRNA與犏牛雄性不育

lncRNA長度一般大于200 nt，主要與染色質(zhì)修飾蛋白、RNA結(jié)合蛋白、小RNA和其他lncRNA相互作用調(diào)節(jié)各種生理過程。lncRNA的功能大致可分為3種調(diào)控模式：競爭者(competitor)、激活者(activator)和前體(precursor)[72]。首先，作為競爭者，lncRNAs可以與某些DNA結(jié)合蛋白結(jié)合，從而抑制其與靶DNA的結(jié)合[73]。例如，一些lncRNAs可以與DNA甲基轉(zhuǎn)移酶1 (DNA methyltransferase 1，DNMT1)結(jié)合，從而阻止DNMT1與靶DNA結(jié)合[74]。因此，DNA靶區(qū)域的甲基化狀態(tài)會(huì)受到影響，導(dǎo)致靶基因的轉(zhuǎn)錄激活。其次，與競爭者相反，lncRNAs還可以作為招募者，通過將表觀遺傳修飾因子招募到特定的靶位點(diǎn)來啟動(dòng)表觀遺傳修飾，從而加強(qiáng)DNA甲基化或組蛋白修飾[75,76]。第三，lncRNAs可以被某些核糖核酸酶(RNase)如Dicer消化，產(chǎn)生小的非編碼RNA，如。是一種附睪特異的1.6 kb mRNA樣前體，能產(chǎn)生類似miRNA的小RNA，通過形成類似miRNA的小RNA，下調(diào)CES7/CES5A(carboxylesterase 5A)蛋白的表達(dá)，從而影響精子獲能[77]。在沉默牛的lncRNA(H19母系印跡表達(dá)轉(zhuǎn)錄本)后，生精小管中的細(xì)胞數(shù)量有減少的趨勢(shì)，這影響了IGF-1R (insulin-like growth factor I receptor)在支持細(xì)胞和生精細(xì)胞中的表達(dá)。IGF-1維持多種類型干細(xì)胞的存活，在雄性生殖系中具有重要功能[78]。近期，阿果約達(dá)等[79]通過RNA-seq技術(shù)從犏牛、牦牛和普通牛中篩選出6178個(gè)差異顯著lncRNA轉(zhuǎn)錄本，候選靶基因2676個(gè)，最終篩選出犏牛不育相關(guān)基因(prostaglandin D2 synthase)、(insulin like growth factor 2和(mesoderm specific transcript)等，這些差異靶基因與犏牛雄性不育相關(guān)。伍仕鑫[80]通過分離出犏牛、牦牛睪丸高純度精原細(xì)胞進(jìn)行l(wèi)ncRNA的RNA-seq、GO和KEGG富集分析后，發(fā)現(xiàn)差異lncRNAs的靶基因(Cyclin-dependent kinase 1)、(BRCA1 DNA Repair Associated)、(Claspin)和(G2/mitotic-specific cyclin-B1)等主要參與犏牛生精過程中細(xì)胞分裂的起始(圖1)，細(xì)胞周期負(fù)性調(diào)控和相變調(diào)控，細(xì)胞周期進(jìn)程的檢控，DNA復(fù)制的損傷檢測及修復(fù)，同源重組和細(xì)胞的內(nèi)吞、程序性死亡、生長、分化和凋亡，以及細(xì)胞物質(zhì)代謝等重要的信號(hào)通路和生物學(xué)過程。這些結(jié)果提示lncRNA可以作為犏牛雄性不育的重點(diǎn)研究方向。

2.3 組蛋白甲基化與犏牛雄性不育

組蛋白甲基化是發(fā)生在精氨酸和賴氨酸上的共價(jià)修飾，是影響基因活性的一種表觀遺傳機(jī)制，其功能主要體現(xiàn)在異染色質(zhì)形成、基因印記、X染色體失活和轉(zhuǎn)錄調(diào)控等方面[81]。它由組蛋白甲基轉(zhuǎn)移酶(histone methyltransferase, HMT)調(diào)節(jié)，可在精氨酸和賴氨酸殘基中添加或移除甲基[82,83]，所以組蛋白賴氨酸甲基化修飾與基因的活化或抑制有關(guān)。通常認(rèn)為，組蛋白H3K4﹑H3K36和H3K79的甲基化與轉(zhuǎn)錄活化基因有關(guān)，而H3K9﹑H3K27和H4K20的甲基化抑制基因表達(dá)[84~86]。例如MLL5/KMT2E (lysine methyltransferase 2E)催化組蛋白H3K4二甲基化(H3K4me2)，是形成頂體所必須的組蛋白甲基轉(zhuǎn)移酶，基因敲除的小鼠是不育的[87]。而H3K9的去甲基化在減數(shù)分裂末期對(duì)于精子發(fā)生的完成至關(guān)重要，否則會(huì)抑制魚精蛋白1 (protamine 1, PRM1)和過渡性蛋白1 (transition protein 1, TNP1)表達(dá)，進(jìn)而導(dǎo)致染色質(zhì)凝集和不育[88]。由此可見，組蛋白甲基轉(zhuǎn)移酶在精子發(fā)生中發(fā)揮著重要作用。犏牛支持細(xì)胞中組蛋白H3K4三甲基化(H3K4me3)缺失，H3K27me3和H4K20me3顯著富集(圖1)，H3K4me3、H3K9me1、H3K9me3和H4K20me3在犏牛精母細(xì)胞減數(shù)分裂染色體中的水平和定位存在顯著差異，這些結(jié)果提示了組蛋白甲基化在精子發(fā)生和犏牛雄性不育中的潛在作用[89]。

3 結(jié)語與展望

近年來分子生物學(xué)興起，特別是組學(xué)技術(shù)的發(fā)展，可以從全基因組水平分析基因表達(dá)、組蛋白甲基化、DNA甲基化和非編碼RNA等與犏牛雄性不育的關(guān)系，為理解犏牛雄性不育的分子機(jī)理提供了新的角度與見解。但要闡述犏牛雄性不育的分子機(jī)制，以下幾個(gè)方面需要引起特別注意：(1)由于睪丸組織細(xì)胞異質(zhì)性和精子發(fā)生的動(dòng)態(tài)與連續(xù)性，基于睪丸組織的組學(xué)研究難以明確具體發(fā)生紊亂的細(xì)胞類型及發(fā)育階段；(2)由于精子發(fā)生細(xì)胞無法建立體外培養(yǎng)體系，對(duì)候選基因的功能驗(yàn)證需要借助小鼠敲除/敲入體系；(3)前期研究主要關(guān)注生精相關(guān)細(xì)胞的表達(dá)調(diào)控關(guān)系，忽略了支持細(xì)胞等睪丸微環(huán)境對(duì)精子發(fā)生的調(diào)控作用[64,89]；(4)各種表觀遺傳因素表現(xiàn)出細(xì)胞類型特異性和生精階段特異性的表達(dá)調(diào)控特性，闡述不同表觀遺傳因素的動(dòng)態(tài)調(diào)控網(wǎng)絡(luò)是今后一個(gè)主要方向，如piRNAs主要在哺乳動(dòng)物粗線期精母細(xì)胞表達(dá)[29,58]，miRNA參與調(diào)控支持細(xì)胞和精原細(xì)胞[64,69]。

綜上所述，表觀遺傳調(diào)控在犏牛雄性不育過程中起到極為關(guān)鍵的作用。今后研究可重點(diǎn)關(guān)注犏牛睪丸支持細(xì)胞和粗線期以前階段生精細(xì)胞，DNA甲基化、非編碼RNA和組蛋白甲基化等表觀遺傳角度是研究犏牛雄性不育的良好切入點(diǎn)。

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Progress on meiotic gene expression and epigenetic regulation of male sterility in Dzo cattle

Huiyou Chen, Jianmin Zhang, Baisen Li, Yonglin Deng, Gongwei Zhang

Interspecific hybrid male sterility is a common occurrence in nature and plays an important role in species reproductive isolation. Dzo (cattle-yak), the offspring of interspecific cross between domestic yak () and cattle (), is a unique animal model for investigating interspecific hybrid male sterility. Dzo females are completely fertile while the males are sterile. In recent years, molecular studies have demonstrated that the expressions of genes were dysregulated during meiosis in Dzo testis, as compared to those in cattle or yak. Other studies have revealed that epigenetic factors/events, such as DNA methylation, histone modification and non-coding RNA, are also involved in spermatogenesis. This review summarizes the dysregulation of gene expression, DNA methylation, microRNA (miRNA), PIWI-interacting RNA (piRNA), long non-coding RNA (lncRNA), and histone methylation modification during meiosis in Dzo testis. These results highlighted the potential roles of genetic and epigenetic regulations of meiosis in Dzo testis, thereby providing a more detailed understanding on the molecular mechanisms of interspecific hybrid male sterility.

Dzo; male sterility; epigenetic; gene expression

2020-06-15;

2020-07-14

國家自然科學(xué)基金項(xiàng)目(編號(hào)：31802046)和中央高?；究蒲袠I(yè)務(wù)費(fèi)(編號(hào)：XDJK2020D011，XDJK2019RC001)資助[Supported by the National Natural Science Foundation of China (No. 31802046) and the Fundamental Research Funds for the Central Universities (Nos. XDJK2020D011, XDJK2019RC001)]

陳會(huì)友，在讀碩士研究生，專業(yè)方向：動(dòng)物遺傳育種。E-mail: 17602359737@163.com

張龔煒，博士，副教授，碩士生導(dǎo)師，研究方向：動(dòng)物遺傳育種。E-mail: zgw-vip@163.com

10.16288/j.yczz.20-176

https://kns.cnki.net/kcms/detail/11.1913.R.20200929.1343.001.html

URI: 2020/9/30 13:13:34

(責(zé)任編委: 趙要風(fēng))