陳瑞，于帥，陳曉旭，杜健，朱振東，潘傳英，曾文先

（1西北農(nóng)林科技大學(xué)創(chuàng)新實(shí)驗(yàn)學(xué)院，陜西楊凌 712100；2西北農(nóng)林科技大學(xué)動(dòng)物科技學(xué)院，陜西楊凌 712100）

非編碼RNA對(duì)哺乳動(dòng)物精子發(fā)生過(guò)程的調(diào)控

陳瑞1,2，于帥2，陳曉旭2，杜健2，朱振東2，潘傳英2，曾文先2

（1西北農(nóng)林科技大學(xué)創(chuàng)新實(shí)驗(yàn)學(xué)院，陜西楊凌 712100；2西北農(nóng)林科技大學(xué)動(dòng)物科技學(xué)院，陜西楊凌 712100）

精子發(fā)生始于精原干細(xì)胞（spermatogonial stem cells, SSCs），SSCs一部分自我更新，另一部分首先分裂形成Asingle（As）型精原細(xì)胞，進(jìn)而形成Aparied（Apr）型精原細(xì)胞和Aaligned（Aal）型精原細(xì)胞；隨后，Aal型精原細(xì)胞再發(fā)育為A1-A4型精原細(xì)胞、中間型精原細(xì)胞以及B型精原細(xì)胞；B型精原細(xì)胞有絲分裂可形成初級(jí)精母細(xì)胞，經(jīng)歷前細(xì)線期、細(xì)線期、偶線期、粗線期，再經(jīng)減數(shù)分裂形成次級(jí)精母細(xì)胞；當(dāng)圓形精子細(xì)胞形成之后，則經(jīng)細(xì)胞核濃縮等過(guò)程形成晚期的細(xì)長(zhǎng)型成熟精子，隨之最終變形成為精子。這一復(fù)雜的生理過(guò)程需要相關(guān)基因的適時(shí)表達(dá)，并受到轉(zhuǎn)錄和轉(zhuǎn)錄后水平的調(diào)控。研究表明，多種類型的非編碼RNA (ncRNAs)在精子發(fā)生過(guò)程中發(fā)揮著重要作用。ncRNAs包括微小RNAs (miRNAs)、與Piwi蛋白相互作用的RNAs (piRNAs)、長(zhǎng)鏈非編碼RNAs (lncRNAs)、環(huán)狀RNAs（circRNAs）以及內(nèi)源性小干擾RNAs (endo-siRNAs)等。這些ncRNAs的表達(dá)具有細(xì)胞組織特異性和發(fā)育階段特異性，可從時(shí)間和空間上精確調(diào)控精子發(fā)生的整個(gè)過(guò)程。miRNAs是一類長(zhǎng)約 21—25 nt的內(nèi)源性非編碼單鏈RNA分子，廣泛存在于各種生物中，其形成至少需要Drosha和Dicer等兩種RNA酶的參與，可降解靶mRNA或抑制靶mRNA翻譯，對(duì)SSCs干性的維持、自我更新和分化的調(diào)控以及生殖細(xì)胞減數(shù)分裂和精子發(fā)生過(guò)程具有重要的調(diào)控作用。此外，精子發(fā)生過(guò)程中，在生殖細(xì)胞不同階段所表達(dá)的基因也可調(diào)控miRNAs的生成加工過(guò)程。piRNAs是2006年發(fā)現(xiàn)的一種新的小RNA，長(zhǎng)度約24—32nt，其作用與Dicer酶無(wú)關(guān)，能夠與生殖細(xì)胞特異性蛋白Piwi蛋白家族成員結(jié)合，進(jìn)而行使生物學(xué)功能，其主要表現(xiàn)為：在表觀遺傳水平和轉(zhuǎn)錄后水平沉默轉(zhuǎn)座子、反轉(zhuǎn)座子等基因組移動(dòng)遺傳元件，維持生殖細(xì)胞自身基因組穩(wěn)定性和完整性，調(diào)控生殖細(xì)胞增殖、減數(shù)分裂及精子發(fā)生過(guò)程。LncRNAs是一類長(zhǎng)度大于200 nt的ncRNAs，其生成加工過(guò)程與mRNA類似，并且與mRNA有著相似的結(jié)構(gòu)。不同來(lái)源的lncRNAs可通過(guò)轉(zhuǎn)錄前與轉(zhuǎn)錄后多種機(jī)制進(jìn)而調(diào)控SSCs的干性及分化，并且調(diào)控生殖細(xì)胞凋亡。有些lncRNAs還可調(diào)控miRNAs的表達(dá)，進(jìn)而調(diào)控精子發(fā)生過(guò)程。circRNAs是區(qū)別于傳統(tǒng)線性RNA的一類新型 RNA，在不同物種中具有保守性，在組織及不同發(fā)育階段呈特異性表達(dá)。其生成加工方式與其序列相關(guān)，同一基因位點(diǎn)可通過(guò)選擇性環(huán)化產(chǎn)生多種circRNAs進(jìn)而發(fā)揮功能。研究表明，circRNAs可結(jié)合miRNAs從而調(diào)控生精過(guò)程。相對(duì)于其他ncRNAs，endo-siRNAs的生成加工方式更為簡(jiǎn)單，并有著與miRNAs相同的作用方式，在精子發(fā)生和雄性生殖中扮演著重要角色。文中結(jié)合最新的研究進(jìn)展，綜述了幾種ncRNAs的生成及其在精子發(fā)生過(guò)程中的調(diào)控作用，旨在為精子發(fā)生過(guò)程中ncRNAs的進(jìn)一步研究提供參考。

精子發(fā)生；非編碼RNA (ncRNAs)；調(diào)控作用

精子發(fā)生是一個(gè)復(fù)雜的生理過(guò)程，包括有絲分裂、減數(shù)分裂、精子的形成與成熟[1]。該過(guò)程需要相關(guān)基因的適時(shí)表達(dá)，并受到轉(zhuǎn)錄和轉(zhuǎn)錄后水平的調(diào)控[2]。研究發(fā)現(xiàn)，超過(guò)1 000種編碼蛋白的基因在精子發(fā)生中發(fā)揮作用[3-7]。然而，這些基因轉(zhuǎn)錄和翻譯調(diào)控的分子機(jī)制尚不清楚。研究表明，多種類型的非編碼RNA（ncRNAs）在睪丸發(fā)育和精子發(fā)生過(guò)程中發(fā)揮著重要作用。這些ncRNAs的表達(dá)具有細(xì)胞組織特異性和發(fā)育階段特異性，可參與精子發(fā)生過(guò)程中生殖細(xì)胞分化的調(diào)控。ncRNAs包括微小RNAs（miRNAs），與Piwi蛋白相互作用的RNAs（piRNAs），長(zhǎng)鏈非編碼RNAs（lncRNAs），環(huán)狀RNAs（circRNAs）和內(nèi)源性小干擾RNAs (endo-siRNAs)等。目前，關(guān)于ncRNAs 在精子發(fā)生過(guò)程中的研究主要集中在miRNAs和piRNAs，新近有文獻(xiàn)報(bào)道lncRNAs，circRNAs和endo-siRNAs也參與精子發(fā)生的調(diào)控。本文結(jié)合最新的研究進(jìn)展，對(duì) miRNAs、piRNAs、lncRNAs、circRNAs、endo-siRNAs的生成及其對(duì)精子發(fā)生的調(diào)控作用進(jìn)行綜述，以期為ncRNAs對(duì)大家畜精子發(fā)生的調(diào)控研究積累科學(xué)資料。

1 精子發(fā)生

精子發(fā)生是繁衍后代的基礎(chǔ)，不同物種的精子發(fā)生周期不盡相同，豬的精子發(fā)生周期一般為40 d，綿羊?yàn)?0 d，山羊?yàn)?0 d，牛為54—60 d，小鼠為35 d，而人則需要72 d[8]。精子的發(fā)生位于成年雄性動(dòng)物睪丸的曲細(xì)精管中，是一個(gè)精確、復(fù)雜、高效的過(guò)程，主要包括3個(gè)階段[1]：第一階段為精原細(xì)胞的增殖階段，該階段中精原干細(xì)胞（spermatogonial stem cells，SSCs）經(jīng)多次有絲分裂形成大量的精原細(xì)胞。小鼠SSCs 約 經(jīng) 歷 A-single(As)， A-paired(Apr)，A-aligned(Aal)，A1型，A2型，A3型，A4型，中間型及B型精原細(xì)胞這8次有絲分裂，最終形成前細(xì)線期的精母細(xì)胞。隨后，初級(jí)精母細(xì)胞開(kāi)始DNA合成過(guò)程。第二階段為精母細(xì)胞的減數(shù)分裂階段，在該階段，前細(xì)線期精母細(xì)胞穿過(guò)血睪屏障并向近腔室方向移動(dòng)，同時(shí)起始減數(shù)分裂，產(chǎn)生單倍體生精細(xì)胞（又稱精子細(xì)胞）。第三階段為精子細(xì)胞的變形階段，圓形精子細(xì)胞經(jīng)過(guò)復(fù)雜的變化轉(zhuǎn)變?yōu)榫哂屑?xì)長(zhǎng)尾巴及運(yùn)動(dòng)能力的精子。

精子發(fā)生的每一個(gè)階段都受到多種因素的精密調(diào)控，在這些調(diào)控因素中，表觀遺傳修飾發(fā)揮著至關(guān)重要的作用。精子中的表觀遺傳修飾包括 ncRNAs，組蛋白修飾，DNA甲基化，基因組印記，X染色體失活，表觀隔代遺傳與其他涉及染色質(zhì)重塑的調(diào)節(jié)機(jī)制。在雄性動(dòng)物精子發(fā)生過(guò)程中存在著大量的表觀調(diào)控因子，其中一部分調(diào)控因子的功能及機(jī)制已被闡明。例如，精子DNA的異常組裝會(huì)導(dǎo)致雄性小鼠不育，這可能是由于參與DNA重構(gòu)的相關(guān)蛋白調(diào)控異常所導(dǎo)致的[9]。YAN等研究發(fā)現(xiàn)，H1LS（spermatid-specific linker histone H1-like protein）是精子細(xì)胞中特異存在的組蛋白H1的相似蛋白，其參與精子發(fā)生過(guò)程中染色質(zhì)的重塑[10]。MARTIANOV 等在圓形精子細(xì)胞中發(fā)現(xiàn)了H1T2（histone H1 variant）的存在，其在染色質(zhì)濃縮過(guò)程中發(fā)揮作用[11]。研究表明，魚(yú)精蛋白的不正常表達(dá)會(huì)降低雄性動(dòng)物的精液質(zhì)量，從而引發(fā)生殖能力的減弱[12]。基因組印記則保證了父本和母本一方的基因在印跡位點(diǎn)的正確表達(dá)，這個(gè)過(guò)程主要是由DNA甲基化修飾調(diào)節(jié)的。除此之外，ncRNAs對(duì)精子發(fā)生也起著關(guān)鍵作用。

2 ncRNAs與精子發(fā)生

ncRNAs在雄性生殖發(fā)育過(guò)程中發(fā)揮著重要作用，從時(shí)間和空間上精確調(diào)控著精子發(fā)生的整個(gè)過(guò)程。研究表明，其在性別分化、雄性性行為以及生殖細(xì)胞發(fā)育過(guò)程中不可或缺，此外，ncRNAs對(duì)生殖干細(xì)胞干性維持、精原細(xì)胞分化及精母細(xì)胞減數(shù)分裂過(guò)程發(fā)揮著重要的調(diào)控作用。

2.1 miRNAs與精子發(fā)生

2.1.1 miRNAs的形成和作用機(jī)制 miRNAs是一類長(zhǎng)約 21—25 nt的內(nèi)源性非編碼單鏈RNA分子，具有高度保守性、時(shí)序性和組織特異性，對(duì)轉(zhuǎn)錄和轉(zhuǎn)錄后的基因表達(dá)調(diào)控起關(guān)鍵作用。miRNAs的形成至少需要Drosha和Dicer等兩種RNA酶的參與。絕大多數(shù)miRNA可在RNA聚合酶Ⅱ的作用下形成長(zhǎng)的莖環(huán)結(jié)構(gòu)，即初級(jí)miRNA（primary miRNA，pri-miRNA），隨后被定位于細(xì)胞核中的Drosha-DGCR8復(fù)合體所剪切，釋放出長(zhǎng)度約 70 nt的發(fā)夾狀 RNA，成為前體miRNA（precursor miRNA，pre-miRNA）[13-15]。 pre-miRNA在輸出蛋白 Exportin-5（Exp5）的作用下從細(xì)胞核轉(zhuǎn)運(yùn)至細(xì)胞質(zhì)中[16-17]，并在胞質(zhì)中被 Dicer剪切產(chǎn)生約為22 nt的miRNA雙鏈，最終miRNA的雙鏈解鏈形成成熟的miRNA[18]。成熟的miRNA與沉默復(fù)合體（RNA-induced silencing comlex，RISC）結(jié)合，形成miRNP識(shí)別靶基因從而發(fā)揮作用。

大量研究證明, miRNA可降解靶mRNA或抑制靶mRNA翻譯。進(jìn)入RISC復(fù)合體的miRNA，如果miRNA與靶mRNA匹配完全，RISC則降解mRNA；若miRNA與靶基因mRNA 的3’UTR序列不完全配對(duì), 則抑制靶基因mRNA的翻譯來(lái)沉默特定基因[19-20]。

2.1.2 miRNAs在真核模式動(dòng)物精子發(fā)生中的作用1993年，LEE等在線蟲(chóng)中發(fā)現(xiàn)了第一個(gè) miRNA—Lin-4基因[21]。2000年，REINHART等在果蠅中發(fā)現(xiàn)了另一個(gè)miRNA—Let7基因及其靶基因lin-4[22]。此后，人們便廣泛關(guān)注這一 RNA分子。在果蠅中，miR-124作用可產(chǎn)生異常激素，并導(dǎo)致miR-124缺失突變體的雄性果蠅減少與雌性果蠅的交配率，甚至出現(xiàn)了雄-雄求偶的現(xiàn)象。與此同時(shí)，雌性果蠅與野生型雄果蠅交配表現(xiàn)出的渴望遠(yuǎn)遠(yuǎn)超過(guò)與miR-124缺失突變體的交配[23]。盡管miR-124突變體表現(xiàn)出更少的交配，但當(dāng)只有miR-124突變體存在時(shí)仍可成功交配，表明其在自然競(jìng)爭(zhēng)環(huán)境中處于劣勢(shì)地位。最新研究表明，如果在成年果蠅體內(nèi)去除miRNA，則會(huì)導(dǎo)致不育，同時(shí)，這些果蠅開(kāi)始產(chǎn)生雄性和雌性兩種性別決定因子。從某種意義上說(shuō)，一旦它們失去了這種miR-124，果蠅就變成了雌雄雙性體，提示，即使在動(dòng)物長(zhǎng)大成熟以后，miRNAs對(duì)于性別決定也必不可少，它們可以發(fā)送信號(hào)讓卵子和精子發(fā)育，從而保證動(dòng)物的生育能力[24]。這些研究表明，miRNAs對(duì)雄性的性分化和性行為具有重要的調(diào)控作用。

2.1.3 miRNAs在哺乳動(dòng)物精子發(fā)生中的作用miRNAs對(duì)SSCs干性的維持發(fā)揮著重要作用。已有文獻(xiàn)報(bào)道，哺乳動(dòng)物 SSCs可表達(dá) miR-20，miR-21，miR-34c，miR-135a，miR-182，miR-183，miR-146a，miR-204和miR-544[25-28]。在小鼠中，miR-20，miR-21和 miR-106a可參與 SSCs動(dòng)態(tài)平衡的調(diào)控[25，29]。miR-34在山羊SSCs中表達(dá)并促進(jìn)p53依賴性的細(xì)胞凋亡[27]。miR-544可通過(guò)調(diào)節(jié)早幼粒細(xì)胞白血病鋅指基因（promyelocytic leukemia zinc finger，PLZF）進(jìn)而調(diào)節(jié)山羊SSCs的自我更新[30]。此外，有些miRNAs參與精原細(xì)胞分化的調(diào)節(jié)。在維甲酸（retinoic acid，RA）誘導(dǎo)精原細(xì)胞分化時(shí)發(fā)現(xiàn) miR-146[31]，let 7miRNAs家族[32]，miR-17-92和 miR-106b-25[33]表達(dá)的下調(diào)。在細(xì)胞水平上，研究人員比對(duì)了小鼠干細(xì)胞和已分化細(xì)胞的 miRNA的差異表達(dá)，發(fā)現(xiàn)了一些干細(xì)胞特有的miRNA，推測(cè)它們參與細(xì)胞分化過(guò)程，同時(shí)也是維持細(xì)胞干性所必需的。有些miRNAs的表達(dá)具有組織細(xì)胞特異性，表明它們可能參與了分化細(xì)胞的維持[4]。

miRNAs在生殖細(xì)胞減數(shù)分裂和精子發(fā)生過(guò)程中發(fā)揮著重要作用。YAN等[34]利用芯片技術(shù)比較了未成熟與成熟小鼠睪丸中miRNAs的表達(dá)情況，發(fā)現(xiàn)有19種miRNAs在這兩種睪丸中存在顯著的差異表達(dá)，表明這些miRNAs可能對(duì)睪丸的發(fā)育產(chǎn)生影響。研究表明，miRNA-122a主要在雄性生殖細(xì)胞晚期階段表達(dá)，可抑制圓形精子標(biāo)志物的轉(zhuǎn)換蛋白 2（Transition protein 2, TP2）的轉(zhuǎn)錄[35]。miRNA微陣列、RT-PCR或小RNA序列研究證實(shí)，miRNAs高度、廣泛、優(yōu)先的在睪丸和雄性生殖細(xì)胞分化的各階段表達(dá)。YADAV等研究發(fā)現(xiàn)，SSCs、精原細(xì)胞、精母細(xì)胞和精子中會(huì)表達(dá)幾種相同的miRNAs，如miR-34c既存在于 SSCs中，調(diào)控其狀態(tài)，又在精母細(xì)胞和圓形精子細(xì)胞中表達(dá)，在精子發(fā)生后期發(fā)揮重要作用[36]；但有一些 miRNAs只在特定的細(xì)胞類型中表達(dá)。miR-17-92簇通過(guò)下調(diào)E2f1防止生殖細(xì)胞在減數(shù)分裂期發(fā)生凋亡[37]。miR-18在精母細(xì)胞中高表達(dá)，并作用于雄性小鼠生殖細(xì)胞發(fā)育的調(diào)控因子Hsf2，從而調(diào)控精子的發(fā)生過(guò)程[38]。最新研究表明，miR-449在睪丸發(fā)育和成年小鼠精子發(fā)生減數(shù)分裂起始時(shí)期高表達(dá)，其表達(dá)模式與miR-34b/c在精子發(fā)生過(guò)程中類似。WU等發(fā)現(xiàn)miR-449或miR-34位點(diǎn)的突變不會(huì)引起雄性小鼠生殖表型的改變，然而miR-449和miR-34的失活會(huì)導(dǎo)致不育[39]。此外，miR-34c還高表達(dá)于小鼠粗線期精母細(xì)胞和精子細(xì)胞中，并通過(guò)Atf1調(diào)節(jié)生殖細(xì)胞的活力[40]。Tp和Prm的適時(shí)表達(dá)是精子發(fā)生過(guò)程中染色質(zhì)凝縮的先決條件。miR-469可以靶向抑制粗線期精母細(xì)胞和圓形精子細(xì)胞中Tp和Prm的mRNAs的翻譯[41]，而miR-122a則可介導(dǎo)Tp2的mRNA的降解[35]。

綜上所述，miRNAs在精子發(fā)生中呈差異性表達(dá)，與 SSCs干性的維持、精子生成及生殖細(xì)胞減數(shù)分裂中基因轉(zhuǎn)錄后的調(diào)控關(guān)系密切，在生殖系統(tǒng)中起到重要的調(diào)節(jié)作用。

2.2 piRNAs與精子發(fā)生

2.2.1 piRNAs的形成和作用機(jī)制 piRNAs是指與生殖細(xì)胞特異性 Piwi蛋白家族成員相結(jié)合才能發(fā)揮作用的RNA[42]，具有調(diào)控基因沉默和維持基因組穩(wěn)定的功能。piRNAs在抑制轉(zhuǎn)座子活性和維持基因組穩(wěn)定性方面起重要作用，但其發(fā)生和調(diào)控的分子機(jī)制仍不清楚。果蠅生殖細(xì)胞為研究這一機(jī)制提供了良好的模型。果蠅生殖細(xì)胞中piRNAs的發(fā)生包括兩種途徑：初級(jí)加工途徑（primary processing pathway）和“乒乓循環(huán)”擴(kuò)增途徑（Ping-Pong amplification loop）。大多數(shù)piRNA序列都對(duì)應(yīng)于范圍較小的基因組區(qū)域，這些區(qū)域被稱為piRNA簇。這些piRNA 簇經(jīng)轉(zhuǎn)錄可得到長(zhǎng)單鏈piRNA前體，隨后經(jīng)過(guò)不依賴于Dicer酶的加工機(jī)制，生成初級(jí)piRNA。在成熟之后，piRNAs與Piwi蛋白相互作用形成piRNA沉默誘導(dǎo)復(fù)合物（piRISCs），介導(dǎo)piRISCs通過(guò)RNA-RNA堿基互補(bǔ)配對(duì)靶向結(jié)合轉(zhuǎn)錄本。已有的假說(shuō)表明，在乒乓循環(huán)的過(guò)程中，Piwi家族成員Aub和Ago3通過(guò)piRNAs識(shí)別的靶RNA進(jìn)行靶向剪切，使得piRNAs得到次級(jí)循環(huán)擴(kuò)增[43]。

2.2.2 piRNAs在真核模式動(dòng)物精子發(fā)生中的作用Piwi蛋白是Argonautue（Ago）蛋白家族的一個(gè)分支，首次在果蠅中發(fā)現(xiàn)，對(duì)生殖干細(xì)胞干性的維持起著重要作用[44]。果蠅中，已經(jīng)鑒定出5種Ago蛋白：Ago1，Ago2，Ago3，Piwi和Aubergine（Aub）[45]。Ago3，Piwi和Aub與非編碼小RNA的另一個(gè)成員：重復(fù)相關(guān)小RNA（repeat associated small interfering RNAs，rasiRNAs）也存在相互作用關(guān)系[46]。rasiRNA最初發(fā)現(xiàn)于胚胎期的果蠅和斑馬魚(yú)中[47-48]，之后，又在果蠅的生殖細(xì)胞中檢測(cè)到了rasiRNA的存在。rasiRNA與Piwi蛋白相結(jié)合可能在生殖細(xì)胞中沉默逆轉(zhuǎn)錄轉(zhuǎn)座子和重復(fù)元件來(lái)調(diào)節(jié)果蠅生殖系的發(fā)育，它的沉默機(jī)制可能與piRNA的調(diào)節(jié)方式存在一定的關(guān)系[49]。

Piwi亞家族主要有3個(gè)成員：Miwi，Mili和Miwi2。果蠅Piwi在生殖細(xì)胞和支持細(xì)胞的細(xì)胞核中表達(dá)，而Miwi和Mili卻存在于胞質(zhì)中。此外，果蠅Piwi突變不但導(dǎo)致精子發(fā)生障礙，同時(shí)也對(duì)生殖細(xì)胞的維持產(chǎn)生影響。Piwi突變的果蠅在小RNA依賴性的轉(zhuǎn)基因和逆轉(zhuǎn)座子沉默上存在缺陷，同時(shí)喪失了異染色質(zhì)蛋白[50]。表明，piRNA能夠沉默轉(zhuǎn)座子，并能防止DNA受損。隨后，研究人員通過(guò)功能缺失突變?cè)囼?yàn)發(fā)現(xiàn)，Piwi的缺失也會(huì)導(dǎo)致線蟲(chóng)生殖細(xì)胞發(fā)育受阻[51]。果蠅的Aub可能與Miwi和Mili更具同源性，因?yàn)锳ub存在于精原細(xì)胞和精母細(xì)胞的細(xì)胞質(zhì)中，Aub功能的缺失會(huì)導(dǎo)致精母細(xì)胞和圓形精子細(xì)胞的不正常發(fā)育。最新研究表明，piRNA可調(diào)控性別決定基因Bmdsx的表達(dá)，在家蠶性別決定過(guò)程中發(fā)揮關(guān)鍵作用[52]。斑馬魚(yú)中兩個(gè)已知的Piwi蛋白是Ziwi和Zili，其中Ziwi在雄性和雌性個(gè)體中都有表達(dá)。Ziwi在斑馬魚(yú)中作為一個(gè)胞內(nèi)蛋白，可能兼有Ago3和Aub的功能。Ziwi水平的降低會(huì)導(dǎo)致生殖細(xì)胞發(fā)生凋亡[53]。有趣的是，Ziwi在斑馬魚(yú)中也可決定性別發(fā)展方向，這表明piRNA可能是性別決定調(diào)控的重要因素。

2.2.3 piRNAs在哺乳動(dòng)物精子發(fā)生中的作用piRNA 與 Piwi亞家族蛋白結(jié)合可形成 piRISCs, piRISCs通過(guò)抑制基因轉(zhuǎn)錄后的調(diào)節(jié)及基因在精子發(fā)生過(guò)程中的異常表達(dá), 從而調(diào)控精子發(fā)生。Piwi亞家族蛋白Miwi、Mili和Miwi2等在哺乳動(dòng)物干細(xì)胞自我更新及雄性生殖細(xì)胞發(fā)育過(guò)程中發(fā)揮重要作用[54-55]。哺乳動(dòng)物Miwi、Miwi2、Mili蛋白表達(dá)于生殖細(xì)胞中后期，是小鼠精子發(fā)生所必需的。敲除Miwi、Mili或 Miwi2基因，都會(huì)使精子產(chǎn)生明顯缺陷，導(dǎo)致雄性不育。Mili蛋白存在于精母細(xì)胞胞質(zhì)、圓形精子細(xì)胞擬染色質(zhì)小體和胞質(zhì)中，在翻譯和維持mRNA的穩(wěn)定性方面發(fā)揮作用。在敲除Mili的小鼠中，精子發(fā)生會(huì)停止在粗線期精母細(xì)胞階段[56]。Miwi表達(dá)于粗線期至圓形精子時(shí)期，敲除 Miwi時(shí)，會(huì)使精子發(fā)生停止在圓形精子階段，不能形成長(zhǎng)形精子。Miwi蛋白的失活會(huì)導(dǎo)致精子細(xì)胞時(shí)期 L1轉(zhuǎn)座子的調(diào)節(jié)異常和生精功能障礙[57]。Miwi與piRISCs結(jié)合可形成有脫腺苷作用的復(fù)合體，也稱染色質(zhì)組裝因子1（Caf1），它可以促進(jìn)脫腺苷化和長(zhǎng)形精子中mRNAs的衰退[58]。敲除Miwi2的小鼠在減數(shù)分裂早期產(chǎn)生缺陷，并且生殖細(xì)胞會(huì)隨著年齡的增長(zhǎng)而產(chǎn)生缺失。Miwi2突變體中生殖細(xì)胞表型的損失，證明了小鼠中的Miwi2和果蠅中的 Piwi在維持生殖系和干細(xì)胞時(shí)起著相似的作用[55]。

小鼠基因組中的 Piwi蛋白在雄性生殖細(xì)胞的分化過(guò)程中受時(shí)間和空間上的調(diào)控，可調(diào)控精子發(fā)生。在小鼠雄性生殖細(xì)胞發(fā)育過(guò)程中，在兩個(gè)不同階段表達(dá)的 piRNAs被分別命名為粗線期前期 piRNAs（pre-pachytene piRNAs）和粗線期piRNAs（pachytene piRNAs）[58]。粗線期前期piRNAs富含轉(zhuǎn)座子序列，在精子發(fā)生早期與Miwi2或Mili共表達(dá)，主要參與胎兒及圍產(chǎn)期雄性生殖細(xì)胞DNA的從頭甲基化。而粗線期piRNAs主要由粗線期精母細(xì)胞和減數(shù)分裂后精細(xì)胞的非轉(zhuǎn)座子基因間區(qū)誘導(dǎo)生成，同Miwi結(jié)合，但其在精子分化過(guò)程中逐漸消失，提示，其可能對(duì)減數(shù)分裂時(shí)相順序的精確進(jìn)行具有調(diào)控作用，進(jìn)而確保具有正常功能的精子生成[59]。最新的研究表明，E3泛素化連接酶和后期促進(jìn)復(fù)合物（APC）能使 Miwi蛋白在精子發(fā)生過(guò)程中發(fā)生泛素化修飾，并通過(guò)泛素蛋白酶體途徑降解。此外，這些能夠激活Miwi蛋白降解的 piRNA也能使自身降解，這表明 Miwi與piRNAs在精子發(fā)生中完成了相應(yīng)作用以后，二者之間也存在著一個(gè)正反饋調(diào)控[60]。

由此可見(jiàn)，piRNAs主要參與雄性生殖細(xì)胞基因組穩(wěn)定性的維持，調(diào)節(jié)生殖細(xì)胞自我更新以及減數(shù)分裂過(guò)程，在精子發(fā)生中起沉默轉(zhuǎn)錄元件的作用。

2.3 lncRNAs與精子發(fā)生

2.3.1 lncRNAs的形成和作用機(jī)制 lncRNAs是一組內(nèi)源性、長(zhǎng)度超過(guò)200 nt、缺乏蛋白質(zhì)編碼能力的RNA分子，包括增強(qiáng)子RNA、基因間轉(zhuǎn)錄本以及與其他轉(zhuǎn)錄本同向或反向重疊的轉(zhuǎn)錄本。與mRNA類似，大多數(shù)lncRNAs由RNA聚合酶Ⅱ轉(zhuǎn)錄，經(jīng)可變剪切形成，并通常被多聚腺苷酸化[61]。近年來(lái)的研究發(fā)現(xiàn)lncRNAs可能具有以下幾方面功能：（1）通過(guò)在蛋白編碼基因上游啟動(dòng)子區(qū)發(fā)生轉(zhuǎn)錄，干擾下游基因的表達(dá)。（2）通過(guò)抑制RNA聚合酶II或者介導(dǎo)染色質(zhì)重構(gòu)以及組蛋白修飾，影響下游基因表達(dá)。（3）通過(guò)與蛋白編碼基因的轉(zhuǎn)錄本形成互補(bǔ)雙鏈，進(jìn)而干擾mRNA的剪切，從而產(chǎn)生不同的剪切形式。（4）通過(guò)與蛋白編碼基因的轉(zhuǎn)錄本形成互補(bǔ)雙鏈，進(jìn)一步在Dicer酶作用下產(chǎn)生內(nèi)源性的 siRNA，調(diào)控基因的表達(dá)。（5）通過(guò)結(jié)合到特定蛋白質(zhì)上，調(diào)節(jié)相應(yīng)蛋白的活性。（6）作為結(jié)構(gòu)組分與蛋白質(zhì)形成核酸蛋白質(zhì)復(fù)合體。（7）通過(guò)結(jié)合到特定蛋白上，改變?cè)摰鞍椎陌|(zhì)定位。（8）作為小分子RNA，如miRNA，piRNA的前體分子轉(zhuǎn)錄[62]。

2.3.2 lncRNAs在精子發(fā)生中的作用 lncRNAs在睪丸發(fā)育過(guò)程中發(fā)揮著重要作用。SUN等利用基因芯片技術(shù)分析小鼠出生后睪丸組織的發(fā)育情況，共檢測(cè)到8 265種lncRNAs，其中3 025種lncRNAs存在差異表達(dá)[63]。2013年，LAIHO等在研究7，14，17，21和28 d小鼠睪丸基因表達(dá)量時(shí)發(fā)現(xiàn)，精子發(fā)生階段共有947種lncRNAs的表達(dá)上調(diào)，這些lncRNAs特定的出現(xiàn)在生殖細(xì)胞發(fā)育的不同階段[64]。同年，BAO等利用基因芯片技術(shù)比較了雄性生殖細(xì)胞發(fā)育關(guān)鍵時(shí)期（12.5 dpc 、15.5 dpc、7 dpp、14 dpp、21 dpp、成年）lncRNAs的表達(dá)情況，結(jié)果表明，有數(shù)千種lncRNAs的表達(dá)被上調(diào)或下調(diào)。此外，大多數(shù)lncRNAs與編碼蛋白的基因有關(guān)，這些編碼蛋白的基因表達(dá)水平與lncRNAs相關(guān)聯(lián)，其在基因組中的位置通?？拷黮ncRNAs[65]。另有一些研究人員發(fā)現(xiàn)小鼠中含有3 639種在 A型精原細(xì)胞特定表達(dá)的 lncRNAs，其中，98種表達(dá)于粗線期精母細(xì)胞，166種表達(dá)于圓形精子細(xì)胞[66]。2014年，CHALMEL等發(fā)現(xiàn)lncRNAs會(huì)在鼠類減數(shù)分裂和精子發(fā)生時(shí)期富集[67]。

盡管在睪丸組織和雄性生殖細(xì)胞中鑒定出了數(shù)千種lncRNAs，但只有少數(shù)的幾種知曉功能。筆者將已知功能的 lncRNAs分為位于常染色體上的 lncRNAs和位于性染色體上的lncRNAs兩類：

（1）位于常染色體上的lncRNAs Mrhl（減數(shù)分裂重組熱點(diǎn)位點(diǎn)）RNA是一個(gè)核富集的lncRNAs，位于小鼠第8號(hào)染色體上，長(zhǎng)度為2.4kb。在小鼠精原細(xì)胞的GC-1 Spg細(xì)胞系中沉默Mrhl RNA，會(huì)導(dǎo)致與細(xì)胞粘附、細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)和細(xì)胞發(fā)育及分化相關(guān)基因的表達(dá)發(fā)生紊亂，這些基因大多在Wnt信號(hào)通路中發(fā)揮重要作用。Mrhl RNA通過(guò)與p68的互作在Wnt信號(hào)通路中發(fā)揮負(fù)調(diào)控作用[68]。

HongrES2是位于小鼠5號(hào)和19號(hào)染色體上，長(zhǎng)度為1.6kb的轉(zhuǎn)錄嵌合物，在附睪尾部的特定區(qū)域表達(dá)。它是類似于miRNA的lncRNA，也是mil-HongrES2的前體。mil-HongrES2能夠抑制嚙齒動(dòng)物Ces7的表達(dá)和固醇酯酶的活性。當(dāng)mil-HongrES2過(guò)表達(dá)時(shí)，會(huì)導(dǎo)致精子獲能遲緩。提示，lncRNAs在小鼠的附睪中發(fā)揮著重要作用[69]。

NLC1-C也稱為基因間RNA162（LINC00162），位于人類21號(hào)染色體上。與正常人相比，在不育男性睪丸組織中，精原細(xì)胞和精母細(xì)胞胞質(zhì)中的 NLC1-C含量較低，而細(xì)胞核中含量較高。NLC1-C在細(xì)胞核中抑制miR-320a和miR-383的轉(zhuǎn)錄。同時(shí)，通過(guò)與核仁蛋白的結(jié)合促進(jìn)體外培養(yǎng)的睪丸胚胎癌細(xì)胞增殖。這些結(jié)論表明NLC1-C通過(guò)與RNA結(jié)合蛋白結(jié)合，進(jìn)而在轉(zhuǎn)錄水平上調(diào)節(jié) miRNA的表達(dá)，從而調(diào)節(jié)人類精子發(fā)生過(guò)程[70]。

spga-lncRNAs是精子發(fā)生過(guò)程中特定表達(dá)的lncRNA，包括spga-lncRNA1和spga-lncRNA2。它們是從一組lncRNAs（109個(gè)，都只含一個(gè)外顯子）中被鑒定出來(lái)的，這些lncRNAs在A型精原細(xì)胞、粗線期精母細(xì)胞和圓形精子細(xì)胞中表達(dá)[71]。這兩種 spgalncRNAs被認(rèn)為是A型精原細(xì)胞分化的抑制因子，暗示了它們?cè)赟SCs干性維持方面發(fā)揮著重要作用。

（2）位于性染色體上的lncRNAs Tsx（核苷特異通道形成蛋白）曾被認(rèn)為是蛋白編碼基因，直至2011年，Anguera等人證實(shí)其為lncRNAs的一種。Tsx在粗線期精母細(xì)胞中特異性表達(dá)，在精原細(xì)胞和圓形精子細(xì)胞中不表達(dá)。在小鼠中敲除Tsx基因，會(huì)促進(jìn)更多的粗線期精母細(xì)胞發(fā)生凋亡，這證實(shí)了其在減數(shù)分裂過(guò)程中發(fā)揮著重要功能[72]。

Dmrt1（doublesex and mab-3 related transcription factor 1）蛋白作為一種重要的轉(zhuǎn)錄因子，可促進(jìn)精子和卵子發(fā)生過(guò)程中特異堿性螺旋-環(huán)-螺旋蛋白 1（Sohlh1）的表達(dá)，同時(shí)抑制Stra8（retinoic acid gene 8）的表達(dá)，進(jìn)而促進(jìn)精子發(fā)生。Dmr（Dmrt1-related gene）的轉(zhuǎn)錄產(chǎn)物是lncRNA的一種，它可破壞Dmrt1的編碼區(qū)，并置換Dmrt1的3′UTR區(qū)域，導(dǎo)致Dmrt1蛋白表達(dá)量的降低，進(jìn)而影響精子發(fā)生[73-74]。

lncRNAs雖然很久以前就被發(fā)現(xiàn)，但直到近年才逐漸被關(guān)注。與生殖相關(guān)的lncRNAs主要參與SSCs干性及分化、生殖細(xì)胞減數(shù)分裂的調(diào)控；有些lncRNAs還可調(diào)節(jié)miRNAs的表達(dá)，從而調(diào)控精子發(fā)生過(guò)程。

2.4 circRNAs與精子發(fā)生

2.4.1 circRNAs的形成和作用機(jī)制 環(huán)狀 RNA（circular RNAs，circRNAs），是區(qū)別于傳統(tǒng)線性RNA的一類新型RNA，它不具有5′末端帽子和3′末端poly（A）尾巴，但以共價(jià)鍵形成閉合環(huán)狀結(jié)構(gòu)。circRNAs在不同物種中具有保守性，在組織及不同發(fā)育階段呈特異性表達(dá)。研究表明，circRNAs在多種生物細(xì)胞中廣泛表達(dá)，同一基因位點(diǎn)或許可通過(guò)選擇性環(huán)化產(chǎn)生多種circRNAs，其在轉(zhuǎn)錄或轉(zhuǎn)錄后水平對(duì)基因表達(dá)調(diào)控具有重要作用。假設(shè)產(chǎn)生 circRNAs的基因含有 4個(gè)外顯子，分別命名為exon1、exon2、exon3和exon4，則circRNAs可能產(chǎn)生的4種模型為：（1）內(nèi)含子配對(duì)（intron pairing）驅(qū)動(dòng)的環(huán)化：位于外顯子側(cè)翼的內(nèi)含子之間存在互補(bǔ)序列，其可直接通過(guò)堿基配對(duì)來(lái)誘導(dǎo)環(huán)化，最終產(chǎn)生Exonic circRNA 或ElciRNA。（2）RNA結(jié)合蛋白（RBP）配對(duì)（RBP pairing）驅(qū)動(dòng)的環(huán)化：結(jié)合到外顯子側(cè)翼內(nèi)含子上的 RBP之間相互作用，最終驅(qū)動(dòng)exon2 與exon3首尾連接進(jìn)而環(huán)化。（3）外顯子跳躍（exon skipping）與套索（intra-lariat）驅(qū)動(dòng)的環(huán)化：前體 RNA部分折疊而發(fā)生外顯子跳躍，使exon1的3′端剪接配體與exon4的5′端剪接受體共價(jià)結(jié)合，形成一個(gè)包含exon2及exon3的套索結(jié)構(gòu)，進(jìn)一步環(huán)化產(chǎn)生circRNA。（4）ciRNA的環(huán)化：內(nèi)含子自身環(huán)化，形成ciRNA[75-78]。

2.4.2 circRNAs在精子發(fā)生中的作用 自circRNAs被發(fā)現(xiàn)來(lái)，一些來(lái)源于真核生物基因組的 circRNAs被鑒定出來(lái)。例如，Y染色體性別決定基因（SRY基因），由一個(gè)外顯子組成，在小鼠睪丸組織中高表達(dá)。在發(fā)育早期，其轉(zhuǎn)錄物可作為蛋白質(zhì)合成的模板，并以線性 RNA的形式存在。但在成年睪丸中，它的RNA主要以環(huán)狀的形式存在于細(xì)胞質(zhì)中，且不具翻譯功能。進(jìn)一步研究發(fā)現(xiàn)，基因組序列兩側(cè)的SRY基因外顯子的反向重復(fù)序列可直接轉(zhuǎn)錄成circRNA。HANSEN等研究發(fā)現(xiàn)，SRY基因的環(huán)狀轉(zhuǎn)錄物含有16個(gè)miR-138的結(jié)合位點(diǎn)，其可抑制 miR-138的活性，從而調(diào)控miR-138靶基因的表達(dá)水平[79]。提示，circRNAs可以調(diào)控miRNA進(jìn)而對(duì)精子發(fā)生產(chǎn)生作用。但目前關(guān)于circRNAs的研究還不深入，有待進(jìn)一步研究。

2.5 Endo-siRNAs與精子發(fā)生

Endo-siRNAs首次報(bào)道于模式生物中，例如果蠅[80]和小鼠[81]。與miRNAs不同，endo-siRNAs的生成不需要Drosha-DGCR8復(fù)合物，它們可以由前體細(xì)胞中正義或反義RNA，或者長(zhǎng)發(fā)夾結(jié)構(gòu)的長(zhǎng)雙鏈RNA（dsRNA）加工而成[82]。

2009年，HAN等在秀麗隱桿線蟲(chóng)中發(fā)現(xiàn)了長(zhǎng)為26 nt的endo-siRNAs，稱為26G endo-siRNAs。26 G RNA基因可以調(diào)節(jié)精子發(fā)生和合子發(fā)育過(guò)程。值得關(guān)注的是，試驗(yàn)中發(fā)現(xiàn)了兩個(gè)26G RNAs 亞類，其中，第一類26G RNAs的靶基因在精子發(fā)生過(guò)程中表達(dá)，第二類26G RNAs來(lái)源于母系遺傳，其可在合子發(fā)育過(guò)程中沉默相關(guān)基因的表達(dá)[83]。

2015年，WU等在小鼠細(xì)胞系中條件性敲除Drosha或Dicer，發(fā)現(xiàn)睪丸中endo-siRNAs的合成需要 Dicer的參與，但與 Drosha無(wú)關(guān)[84]。在果蠅中，hpRNAs（hairpin RNA）被認(rèn)為是endo-siRNAs的一種，其在睪丸中高表達(dá)[85]。在小鼠中，endo-siRNAs在胚胎干細(xì)胞中豐富表達(dá)，其次是SSCs，在成熟生殖細(xì)胞中表達(dá)最低[86]。對(duì)原始生殖細(xì)胞、卵母細(xì)胞和受精卵中的短鏈非編碼 RNA（sncRNAs）的功能分析指出，小RNA和小核仁RNA（snoRNAs）都可在原始生殖細(xì)胞中廣泛表達(dá)，但僅持續(xù)片刻便會(huì)被精子中的干擾RNA和卵母細(xì)胞與受精卵中的endo-siRNAs所替代[87]。在小鼠和其他哺乳動(dòng)物中，生殖細(xì)胞特異性蛋白 Dicer可以影響減數(shù)分裂進(jìn)而導(dǎo)致雄性不育、精母細(xì)胞凋亡的增加和缺陷精子的產(chǎn)生。Drosha敲除小鼠會(huì)產(chǎn)生有缺陷的miRNA途徑，但存在完整的 endo-siRNAs途徑。雖然這些研究揭示了 endosiRNAs在哺乳動(dòng)物精子發(fā)生和雄性生殖中的扮演著重要角色[88]，但其在SSCs干性維持、精子變形等過(guò)程是如何發(fā)揮調(diào)控作用的還有待進(jìn)一步研究。

3 展望

睪丸的發(fā)育決定著種群繁殖的質(zhì)量和優(yōu)良種公畜的利用價(jià)值，對(duì)睪丸發(fā)育的研究也從最初的睪丸形態(tài)組織學(xué)觀察，到編碼基因功能的調(diào)控，再到新近的表觀遺傳調(diào)控因子。隨著基因組計(jì)劃的完成，人們對(duì)非編碼RNA認(rèn)識(shí)的不斷深入，這些非編碼RNA可作為重要的表觀遺傳調(diào)控因子，調(diào)控精子發(fā)生過(guò)程，對(duì)精原干細(xì)胞干性維持、生殖細(xì)胞減數(shù)分裂、精原細(xì)胞分化及精母細(xì)胞減數(shù)分裂等過(guò)程發(fā)揮著重要的調(diào)控作用。然而，其研究多數(shù)集中在微小RNAs、與Piwi蛋白相互作用的RNAs和長(zhǎng)鏈非編碼RNAs上，在環(huán)狀RNAs和內(nèi)源性小干擾RNAs上的研究還處于初級(jí)階段。非編碼RNA在精子發(fā)生中的作用多集中在線蟲(chóng)、果蠅、小鼠和人上，在大家畜上的研究較少。相信，隨著組學(xué)時(shí)代的到來(lái)，對(duì)非編碼RNA研究的不斷深入，將有助于大家畜精子發(fā)生的研究，會(huì)為大家畜生精障礙和精子發(fā)生異常治療方法的選擇提供理論依據(jù)。

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（責(zé)任編輯 林鑒非）

Regulatory Role of Noncoding RNAs During Spermatogenesis

CHEN Rui1,2, YU Shuai2, CHEN XiaoXu2, DU Jian2, ZHU ZhenDong2, PAN ChuanYing2, ZENG WenXian2
(1Innovation Experimental College, Northwest A&F University, Yangling 712100, Shaanxi;2College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi)

Spermatogenesis starts with spermatogonial stem cells (SSCs), which possess the ability of self-renewal anddifferentiation. SSCs are capable of differentiation to form Asingle (As) spermatogonia, Apaired (Apr) spermatogonia, Aaligned (Aal) spermatogonia, A1-A4 spermatogonia, intermediate spermatogonia, and B spermatogonia. Type B spermatogonia divide forming the primary spermatocytes, which undergo a long meiosis time to form secondary spermatocytes. Then secondary spermatocytes go through meiosis II to produce round spermatids, which will undergo a series of processes called spermiogenesis containing morphological changes, replacement histone by protamine, nuclear condensation and formation of flagellum. Finally, the mature spermatozoa are released into the lumen. This process requires precise and highly ordered regulation of gene expression at both the transcriptional and posttranscriptional levels. Recent advances in research have revealed that several types of noncoding RNAs (ncRNAs), including microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs) and endogenous small-interfering RNAs (endo-siRNAs), are essential for spermatogenesis. These ncRNAs are expressed in a cell-specific and step-specific manner to participate in the control of spermatogenesis. MiRNAs are a class of endogenous non coding single stranded RNA molecules of about 21-25 nt that widely exist in various kinds of organisms, its formation needs at least two RNA enzymes such as Drosha and Dicer, which can also degrade target mRNA or inhibit target mRNA translation, have an important regulatory role in maintaining the stemness, self-renewal of SSCs, regulating differentiation, and involved germ cell meiosis and spermatogenesis. piRNAs are a large class of small RNAs that are 24-32 nt in length found in 2006, which could execute the biological function through interactions with Piwi proteins without Dicer enzyme, also silence transposons and retroposons at the epigenetic and posttranscriptional levels, maintain the genomic stability and integrity of germ cell, regulate cell proliferation, meiosis and spermatogenesis. LncRNAs are one of ncRNAs longer than 200 nt, their production process and structure are similar to the mRNA. Different sources of lncRNAs could regulate the stemness, differentiation of SSCs, and modulate germ cell apoptosis in a transcriptional and posttranscriptional manner. Some lncRNAs could also regulate the expression of miRNAs thus regulate the process of spermatogenesis. CircRNAs, differs from the traditional linear RNA, is a new type of RNA, which is conserved in different species, and specifically expressed in different tissues and developmental stages. Its formation processing mode is related to its sequence, the same gene locus could produce a variety of circRNAs through selective cyclization. Studies indicated that circRNAs can be combined with miRNAs to regulate spermatogenesis. Compared with other ncRNAs, the biogenesis of endo-siRNAs is simple, and has the same effect as miRNAs, which plays an important role in spermatogenesis and male reproduction. Therefore, this review summarized the regulatory role of ncRNAs during spermatogenesis, which provided insight into the further research on ncRNAs during spermatogenesis.

spermatogenesis; noncoding RNAs; regulatory role

2016-05-25；接受日期：2016-11-23

國(guó)家自然科學(xué)基金（31572401, 31272439）、中國(guó)博士后科學(xué)基金第56批面上資助項(xiàng)目（2014M560809）和陜西省博士后科研項(xiàng)目

聯(lián)系方式：陳瑞，E-mail：chenrui950122@126.com。通信作者潘傳英，E-mail：chuanyingpan@126.com，panyu1980@126.com