凡納濱對(duì)蝦“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控精巢發(fā)育的分子機(jī)制研究

陳康軒, 李詩豪, 李富花

凡納濱對(duì)蝦“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控精巢發(fā)育的分子機(jī)制研究

陳康軒1, 2, 李詩豪1, 李富花1

(1. 中國科學(xué)院海洋研究所 實(shí)驗(yàn)海洋生物學(xué)重點(diǎn)實(shí)驗(yàn)室, 山東 青島 266071; 2. 中國科學(xué)院大學(xué) 北京 100049)

位于甲殼動(dòng)物眼柄的神經(jīng)內(nèi)分泌器官在調(diào)控甲殼動(dòng)物的繁殖過程中發(fā)揮著重要作用。已有證據(jù)表明“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控十足目雄性甲殼動(dòng)物精巢的發(fā)育, 但是該過程的分子機(jī)制仍不清楚。本研究以凡納濱對(duì)蝦()為實(shí)驗(yàn)對(duì)象, 研究了切除雄性對(duì)蝦的單側(cè)眼柄對(duì)促雄性腺內(nèi)胰島素樣促雄性腺素基因()和精巢內(nèi)基因表達(dá)的影響。結(jié)果表明, 切除單側(cè)眼柄后,基因的表達(dá)明顯上調(diào); 比較了眼柄切除前后對(duì)蝦精巢的轉(zhuǎn)錄組數(shù)據(jù), 共有267個(gè)基因的表達(dá)量出現(xiàn)明顯變化。對(duì)精巢內(nèi)差異表達(dá)基因進(jìn)行功能分析發(fā)現(xiàn), 多個(gè)參與性腺發(fā)育和內(nèi)分泌調(diào)控的基因發(fā)生表達(dá)上調(diào), 如()、保幼激素環(huán)氧水解酶(juvenile hormone epoxide hydrolase,)、細(xì)胞色素P450酶系基因以及泛素化系統(tǒng)相關(guān)基因等。對(duì)差異表達(dá)基因進(jìn)行熒光定量PCR驗(yàn)證, 結(jié)果與轉(zhuǎn)錄組結(jié)果一致, 證實(shí)了轉(zhuǎn)錄組結(jié)果的可靠性。研究結(jié)果表明, 對(duì)蝦眼柄調(diào)控精巢的發(fā)育很可能是通過影響促雄性腺中的表達(dá), 進(jìn)而調(diào)控精巢內(nèi)、、內(nèi)分泌及泛素化系統(tǒng)相關(guān)基因的表達(dá)實(shí)現(xiàn)的。研究結(jié)果對(duì)深入理解甲殼動(dòng)物精巢發(fā)育的分子調(diào)控機(jī)制提供了重要依據(jù), 對(duì)甲殼動(dòng)物的人工繁育也具有重要指導(dǎo)意義。

對(duì)蝦; 眼柄; 胰島素樣促雄性腺素; 精巢發(fā)育

十足目甲殼動(dòng)物眼柄內(nèi)的X器官/竇腺復(fù)合體(X-organ/sinus-gland complex, XO-SG)是其神經(jīng)內(nèi)分泌系統(tǒng)的中樞, 在調(diào)節(jié)動(dòng)物新陳代謝、蛻皮、生殖發(fā)育等生理過程發(fā)揮著重要作用[1, 2]。通過人工摘除眼柄, 可以起到促進(jìn)蛻皮、性腺成熟及生長(zhǎng)的作用[3-7]。XO-SG復(fù)合體合成分泌的蛻皮抑制激素(Molt-inhibiting hormone, MIH)、甲殼動(dòng)物雌性性激素(Crustacean female sex hormone, CFSH)、性腺抑制激素(Gonad inhibiting hormone, GIH)、甲殼動(dòng)物高血糖激素(Crustacean hyperglycemic hormone, CHHs)等一系列神經(jīng)肽激素在這些生理過程的調(diào)控中發(fā)揮著重要作用[8-13]。促雄性腺(androgenic gland, AG)是雄性甲殼動(dòng)物所特有的, 其通過分泌胰島素樣促雄性腺素(Insulin-like androgenic gland hormone, IAG)參與甲殼動(dòng)物雄性性別分化過程。雄性甲殼動(dòng)物眼柄中的XO-SG復(fù)合體可以抑制促雄性腺的發(fā)育和基因的表達(dá), 其中已經(jīng)證實(shí)CHH家族等眼柄神經(jīng)肽類激素直接負(fù)調(diào)控基因的表達(dá)[9, 14-17]。研究表明, 雄性甲殼動(dòng)物的性別發(fā)育過程受到“眼柄-促雄性腺-精巢”內(nèi)分泌軸的調(diào)控[14]。然而, 精巢中哪些基因受到“眼柄-促雄性腺-精巢”內(nèi)分泌軸的調(diào)控參與雄性性別發(fā)育目前仍不十分清楚。轉(zhuǎn)錄組技術(shù)的發(fā)展和應(yīng)用實(shí)現(xiàn)了對(duì)特定器官在特定條件下基因表達(dá)水平的高通量分析, 一系列性別發(fā)育相關(guān)基因也得到鑒定, 使得構(gòu)建甲殼動(dòng)物性腺發(fā)育的分子調(diào)節(jié)網(wǎng)絡(luò)成為可能。本研究以凡納濱對(duì)蝦為實(shí)驗(yàn)對(duì)象, 研究了雄性對(duì)蝦的單側(cè)眼柄切除對(duì)促雄性腺內(nèi)基因及精巢內(nèi)基因表達(dá)的影響, 發(fā)現(xiàn)對(duì)蝦基因及精巢內(nèi)多個(gè)參與性腺發(fā)育和內(nèi)分泌調(diào)控的基因發(fā)生明顯上調(diào)表達(dá)。研究結(jié)果為深入理解甲殼動(dòng)物精巢發(fā)育的分子調(diào)控機(jī)制提供了重要依據(jù), 對(duì)甲殼動(dòng)物的人工繁育也具有重要指導(dǎo)意義。

1 材料與方法

1.1 實(shí)驗(yàn)材料

健康的雄性凡納濱對(duì)蝦()來自于青島市附近的養(yǎng)殖場(chǎng)(體長(zhǎng)12 cm±0.2 cm, 體質(zhì)量10.5 g±1.5 g)。

1.2 方法

1.2.1 眼柄切除

將60 尾健康的雄性凡納濱對(duì)蝦分為兩組, 一組使用酒精噴燈燒紅的鑷子從基部燙斷右側(cè)眼柄, 另外一組作為對(duì)照組, 正常飼養(yǎng)。在(25±1) ℃曝氣海水中飼養(yǎng)一周后, 取促雄性腺組織和精巢組織于液氮中速凍, –80 ℃冰箱保存。

1.2.2 總RNA提取及cDNA合成

使用Takara公司的RNAisol方法提取AG及精巢組織的總RNA。使用Nanodrop 2000檢測(cè)樣品濃度與純度, RNA質(zhì)量檢測(cè)使用1.5%的瓊脂糖凝膠電泳方法。使用Thermo公司的Revert Aid Fist Strand cDNA synthesis Kit進(jìn)行AG和精巢組織cDNA第一鏈的合成。

1.2.3基因表達(dá)水平檢測(cè)及轉(zhuǎn)錄組樣品制備

使用熒光定量PCR分別對(duì)眼柄切除組和對(duì)照組每只對(duì)蝦AG組織的基因表達(dá)水平進(jìn)行檢測(cè)。測(cè)定后, 根據(jù)基因表達(dá)水平, 選取實(shí)驗(yàn)組中該基因表達(dá)水平提高幅度較大的9個(gè)個(gè)體的精巢RNA樣品用于轉(zhuǎn)錄組測(cè)序, 另外從對(duì)照組中選擇該基因表達(dá)水平變化不大的9個(gè)體的精巢RNA樣品用于轉(zhuǎn)錄組測(cè)序。為避免蛻皮時(shí)期對(duì)基因表達(dá)的影響, 所取樣對(duì)蝦均處于蛻皮間期。每組選擇的9個(gè)精巢RNA樣品每3個(gè)混合成一個(gè)樣品, 每組包含3個(gè)生物學(xué)重復(fù)樣品。實(shí)驗(yàn)組3個(gè)樣品分別命名為EAT1、EAT2和EAT3, 對(duì)照組3個(gè)樣品分別命名為ECT1、ECT2和ECT3。

1.2.4 轉(zhuǎn)錄組測(cè)序

RNA樣品送至廣州基迪奧生物科技公司測(cè)序, 首先使用帶 Oligo (dT) 的磁珠富集真核生物mRNA, 超聲片段化后作為模板進(jìn)行cDNA雙鏈合成。純化后的雙鏈cDNA經(jīng)過末端修復(fù)與添加poly(A)尾后與測(cè)序接頭連接。用AMPure XP beads篩選200 bp左右的cDNA, 進(jìn)行PCR擴(kuò)增, 最后使用AMPure XP beads純化PCR產(chǎn)物, 獲得測(cè)序文庫。利用瓊脂糖凝膠電泳、Qubit2.0 Fluorometer以及Agilent 2100 bioanalyzer等方法檢測(cè)樣品純度、濃度與完整性后, 符合標(biāo)準(zhǔn)的文庫使用Illumina HiseqTM 2500測(cè)序儀進(jìn)行雙末端測(cè)序。

1.2.5 測(cè)序數(shù)據(jù)比對(duì)

使用短reads比對(duì)工具Bowtie2(version 2.2.8)剔除核糖體RNA(rRNA)序列獲得clean reads。利用凡納濱對(duì)蝦參考基因組(PRJNA438564)為索引, 使用HISAT2.2.4將雙端測(cè)序的clean reads在參考基因組上進(jìn)行比對(duì)。

1.2.6 生物信息分析

轉(zhuǎn)錄組數(shù)據(jù)分析參照基迪奧公司有參轉(zhuǎn)錄組分析的基本流程, 包括數(shù)據(jù)質(zhì)控、序列比對(duì)分析、基因分析、 基因表達(dá)量統(tǒng)計(jì)、樣本關(guān)系分析、組間差異分析、基因功能富集等。差異表達(dá)基因的篩選標(biāo)準(zhǔn)為: 差異倍數(shù)>2和錯(cuò)誤發(fā)現(xiàn)率≤0.01。

通過從相應(yīng)的InterProScan或Pfam結(jié)果中基因本體注釋并進(jìn)行富集分析。使用KEGG自動(dòng)注釋服務(wù)器將差異表達(dá)基因信息與KEGG數(shù)據(jù)庫進(jìn)行比對(duì), 對(duì)涉及的基因通路進(jìn)行注釋。

1.2.7 實(shí)時(shí)熒光定量PCR

對(duì)篩選到的精巢差異表達(dá)基因, 使用Primer3.0 plus設(shè)計(jì)特異性引物(表1), 使用實(shí)時(shí)熒光定量PCR驗(yàn)證基因表達(dá)情況。以眼柄切除前后的精巢RNA反轉(zhuǎn)錄的cDNA為模板, 使用18S rRNA作為內(nèi)參基因, 擴(kuò)增條件為: 95 ℃預(yù)變性4 min; 95 ℃變性15 s, 56 ℃退火 20 s, 72 ℃延伸30 s, 擴(kuò)增40個(gè)循環(huán)。使用2–ΔΔCT法計(jì)算基因的相對(duì)表達(dá)量, 對(duì)數(shù)據(jù)采用單因素方差分析[18]進(jìn)行統(tǒng)計(jì)學(xué)檢驗(yàn), 以< 0.05和<0.01作為顯著性差異和極顯著差異的評(píng)價(jià)標(biāo)準(zhǔn)。

2 結(jié)果

2.1 眼柄切除對(duì)LvIAG基因表達(dá)的影響

通過real-time PCR 法檢測(cè), 眼柄切除組中基因在促雄腺組織中的相對(duì)表達(dá)量與對(duì)照組相比上調(diào)約12.2倍(圖1), 表明眼柄切除后引起了基因的表達(dá)顯著上調(diào)。

表1 引物的序列信息及退火溫度

圖1 眼柄切除處理組與對(duì)照組LvIAG基因在促雄腺中的表達(dá)水平檢測(cè)

**. 差異極顯著(<0.01)

**. very significant difference(<0.01)

2.2 轉(zhuǎn)錄組數(shù)據(jù)分析及差異表達(dá)基因鑒定

轉(zhuǎn)錄組測(cè)序共得到原始測(cè)序序列總計(jì)230 724 552條, 經(jīng)過質(zhì)量控制后獲得高質(zhì)量序列共226 147 040條, 占總測(cè)序數(shù)據(jù)的98.01%, 測(cè)序信息統(tǒng)計(jì)見表2。獲得的測(cè)序數(shù)據(jù)與參考基因組比對(duì)率為83.05%～85.39%, 過濾后Q20范圍為97.74%～ 98.18%, Q30在93.62%～94.44%。以上數(shù)據(jù)說明轉(zhuǎn)錄組測(cè)序的總體質(zhì)量較好, 可用于后續(xù)的差異基因表達(dá)分析。

通過本次精巢差異轉(zhuǎn)錄組測(cè)序, 共獲得267個(gè)差異表達(dá)基因, 其中有238個(gè)基因呈現(xiàn)上調(diào)表達(dá), 29個(gè)基因呈現(xiàn)下調(diào)表達(dá)(圖2)。

2.3 差異表達(dá)基因的富集分析

KEGG通路富集分析結(jié)果顯示, 眼柄切除的對(duì)蝦精巢轉(zhuǎn)錄組中差異表達(dá)基因主要富集在特定的代謝途徑, 包括轉(zhuǎn)運(yùn)與分解代謝(Transport and catabolism)、碳水化合物代謝(carbohydrate metabolism)、信號(hào)分子與互作(signaling molecules and interaction)等(圖3)。

表2 實(shí)驗(yàn)組和對(duì)照組精巢的轉(zhuǎn)錄組測(cè)序數(shù)據(jù)信息

圖2 實(shí)驗(yàn)組和對(duì)照組精巢差異表達(dá)基因的火山圖

注: 紅色點(diǎn): 表達(dá)上調(diào)基因; 黃色點(diǎn): 表達(dá)下調(diào)基因; 藍(lán)色點(diǎn): 表達(dá)差異不顯著基因

使用GO數(shù)據(jù)庫, 將眼柄切除實(shí)驗(yàn)組中鑒定的精巢差異表達(dá)基因按照生物學(xué)過程、細(xì)胞組分和分子功能分別進(jìn)行統(tǒng)計(jì)分析, 共富集到3個(gè)大類的15個(gè)過程中, 結(jié)果如圖4所示。生物過程大類富集最多, 包括代謝過程(metabolism process)、單一生物過程(single- or-ganism process) 以及細(xì)胞學(xué)過程(cellular process)。細(xì)胞組分大類中, 具有結(jié)合(binding)和催化活性(catalytic activity)的基因富集較多。

圖3 實(shí)驗(yàn)組和對(duì)照組對(duì)蝦精巢差異表達(dá)基因KEGG通路富集結(jié)果

注: EAT為眼柄切除實(shí)驗(yàn)組; ECT為對(duì)照組

圖4 對(duì)蝦精巢差異表達(dá)基因GO富集分析

注: 黃色部分: 實(shí)驗(yàn)組相對(duì)對(duì)照組表達(dá)上調(diào)基因; 藍(lán)色部分: 實(shí)驗(yàn)組相對(duì)對(duì)照組表達(dá)下調(diào)基因

2.4 差異表達(dá)基因的功能分類與定量驗(yàn)證

根據(jù)已報(bào)道基因的功能, 將眼柄切除前后精巢中差異表達(dá)基因進(jìn)行分類, 鑒定了一系列與性別發(fā)育、內(nèi)分泌系統(tǒng)和泛素化系統(tǒng)等相關(guān)的基因(表3)。性別發(fā)育相關(guān)的基因表達(dá)量顯著上調(diào)。內(nèi)分泌系統(tǒng)相關(guān)的差異表達(dá)基因均呈現(xiàn)上調(diào)表達(dá), 包括可能參與MF降解的保幼激素酯酶基因和細(xì)胞色素P450酶系基因和。泛素化系統(tǒng)相關(guān)基因包括泛素激活酶E1(ubiquitin-activating enzyme E1)、泛素偶聯(lián)酶E2(ubiquitin-conjugating enzyme E2)和泛素特異性肽酶USP21(ubiquitin specific peptidase 21)均呈現(xiàn)上調(diào)表達(dá)趨勢(shì), 而E3泛素連接酶SH3RF1(E3 ubiquitin-protein ligase SH3RF1)則呈現(xiàn)下調(diào)表達(dá)。

表3 對(duì)蝦精巢差異表達(dá)基因功能分類

為了驗(yàn)證轉(zhuǎn)錄組中所鑒定差異表達(dá)基因的可靠性, 選取了5個(gè)差異表達(dá)基因、、、、進(jìn)行RT-qPCR驗(yàn)證。結(jié)果如圖5所示, 每個(gè)基因的轉(zhuǎn)錄組數(shù)據(jù)與RT-qPCR結(jié)果均具有相同的表達(dá)趨勢(shì), 表明轉(zhuǎn)錄組差異表達(dá)基因的鑒定結(jié)果是可靠的。

圖5 精巢差異表達(dá)基因的轉(zhuǎn)錄組數(shù)據(jù)和RT-qPCR結(jié)果比較

注: *: 差異顯著(<0.05); **: 差異極顯著(<0.01)

3 討論

本研究通過對(duì)雄性凡納濱對(duì)蝦進(jìn)行單側(cè)眼柄切除, 顯著提高了促雄性腺中基因的表達(dá), 表明眼柄切除一定程度上解除了眼柄內(nèi)分泌系統(tǒng)中抑制性別發(fā)育的因素。在此前提下, 通過對(duì)眼柄切除前后對(duì)蝦的精巢進(jìn)行比較轉(zhuǎn)錄組分析, 鑒定到一系列與性別發(fā)育、內(nèi)分泌、泛素化等過程相關(guān)的差異表達(dá)基因, 它們可能受到“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控參與精巢的發(fā)育過程。

3.1 性別發(fā)育相關(guān)基因受“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控參與精巢發(fā)育

促雄性腺表達(dá)分泌的胰島素樣促雄性腺素(IAG)除了具有促進(jìn)雄性甲殼動(dòng)物動(dòng)物性別分化的作用外[19],在維持雄性性征及精子發(fā)生等方面也發(fā)揮關(guān)鍵作用[20]。眼柄切除后,基因表達(dá)量顯著上升, 這與眼柄內(nèi)分泌系統(tǒng)中表達(dá)分泌該基因的抑制因子如CHH等神經(jīng)肽激素密切相關(guān)[9]。眼柄切除引起的基因表達(dá)升高, 對(duì)于精巢的發(fā)育過程具有促進(jìn)作用。

Doublesex()是Dmrt家族成員, 它作為性別級(jí)聯(lián)通路下游的關(guān)鍵調(diào)控因子, 具有調(diào)控性別分化過程的作用[21-23]。在中國明對(duì)蝦()中,基因表達(dá)被沉默后, 顯著降低了基因的表達(dá)水平, 表明對(duì)基因的表達(dá)起到正向調(diào)控作用[24]。同時(shí), 在個(gè)體發(fā)育過程中,基因也具有維持性腺發(fā)育的功能。多種甲殼物種中的基因在成體中呈現(xiàn)出性別二態(tài)性表達(dá)特征[25-27]。部分基因在甲殼動(dòng)物精巢中特異性高表達(dá), 說明其可能參與了精子發(fā)生過程[28-29]。在本研究中, 眼柄切除后的精巢內(nèi)基因表達(dá)量顯著上調(diào), 表明可能在“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控下參與精巢發(fā)育的過程。

3.2 細(xì)胞色素P450酶系基因受“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控參與精巢發(fā)育

在脊椎動(dòng)物中, 合成類固醇的過程需要細(xì)胞色素P450酶系的參與, 例如芳香化酶基因?qū)τ诩棺祫?dòng)物性腺分化和發(fā)育起了重要作用[30]。在節(jié)肢動(dòng)物中, 已有多個(gè)P450家族成員被報(bào)道具有調(diào)節(jié)內(nèi)源性信號(hào)分子(包括蛻皮激素)、信號(hào)分子與防御用途化學(xué)物質(zhì)的合成和降解的功能[31]。在甲殼動(dòng)物中, 細(xì)胞色素P450被認(rèn)為可以合成與代謝各種類固醇激素, 其中蛻皮激素被認(rèn)為是細(xì)胞色素P450最重要的底物[32]。在海洋橈足類飛馬哲水蚤()中, 含有較高脂肪儲(chǔ)備的雌性個(gè)體, 體內(nèi)往往有著高濃度的20E, 而在這類個(gè)體中表達(dá)量最高, 表明其參與蛻皮酮合成和脂質(zhì)儲(chǔ)存調(diào)節(jié)過程[33]。在普通濱蟹()中,也被發(fā)現(xiàn)可能具有調(diào)節(jié)20E合成的作用[34]。

甲殼動(dòng)物性腺和血淋巴中廣泛存在多種類固醇類激素。三疣梭子蟹()的卵巢組織中發(fā)現(xiàn)了17-α羥基化孕酮[35], 美洲龍蝦()精巢中發(fā)現(xiàn)孕酮會(huì)被轉(zhuǎn)化為20-α-羥基孕酮[36], 斑節(jié)對(duì)蝦()卵巢中的孕酮可被代謝為20α-dihydroprogesterone[36]。然而, 類固醇激素在甲殼動(dòng)物發(fā)育、生長(zhǎng)、性別分化以及繁殖等方面的具體作用以及細(xì)胞色素P450是否參與這些類固醇激素合成與代謝都還不明確。本研究發(fā)現(xiàn)的基因, 其同源基因最早在對(duì)加勒比海眼斑龍蝦()中報(bào)道, 對(duì)黃體酮和睪酮的單加氧反應(yīng)具有催化作用[37]。及其他P450家族基因在眼柄切除后的顯著上調(diào), 說明它們可能受“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控, 在凡納濱對(duì)蝦精巢發(fā)育過程中具有重要作用。

3.3 泛素化相關(guān)基因在性腺發(fā)育中的作用

泛素/蛋白酶體途徑(ubiquitin-proteasome system, UPP)在細(xì)胞周期調(diào)控、細(xì)胞器生物發(fā)生、組織重塑等許多生化過程中發(fā)揮著重要作用[38]。泛素化依賴于3個(gè)泛素化酶的協(xié)同作用: 泛素激活酶E1以ATP依賴的方式將泛素分子C端賴氨酸(Lys)殘基同自身半胱氨酸(Cys)以硫酯鍵連接, 之后泛素分子通過高能硫酯鍵連接到泛素偶聯(lián)酶E2, 最終直接連接到目標(biāo)蛋白或在泛素連接酶E3酶作用下與靶蛋白通過形成氨基異肽鍵連接, 從而實(shí)現(xiàn)對(duì)靶蛋白的標(biāo)記分類[39]。泛素化調(diào)節(jié)過程也是可逆的, 這一過程由去泛素化酶(deubiquitinating enzymes, DUBs)介導(dǎo), 主要分為5個(gè)家族: 泛素羧基末端水解酶(ubiquitin carboxy-terminal hydrolases, UCH)家族, 泛素特異性蛋白酶(ubiquitin-specific proteases, USP/ UBP)家族, Otubaim(OTU)家族, Josephin結(jié)構(gòu)域蛋白家族及AB1/MPN/Mov34 metalloenzyme(JAMM)家族[40]。

UPP在性別發(fā)育過程中的作用已在多種動(dòng)物中有報(bào)道。在家豬(Sus scrofa domestica)中,編碼的泛素激活酶E1參與到了精子發(fā)生過程, 在精子獲能和頂體功能方面發(fā)揮作用[41]。在大鼠()中,特異表達(dá)于精原細(xì)胞, 其可能通過泛素/蛋白酶體系統(tǒng)來影響精子發(fā)生[42]。在小鼠()中, 使用特異性抑制劑PYR-41 處理的卵細(xì)胞, 通過干擾常見的泛素化靶蛋白β-catenin, 導(dǎo)致精卵融合后精子體積的增大和染色體減數(shù)分裂的阻滯[43]。在秀麗隱桿線蟲()中, Cul-2泛素連接酶復(fù)合物以Fem-1為識(shí)別亞基, 以Fem-2和Fem-3輔因子, 通過蛋白酶體依賴性降解TRA-1實(shí)現(xiàn)性別決定的最后一步[44]。線蟲基因發(fā)揮重要的泛素化功能, 在精子發(fā)生、體型調(diào)控和性別分化發(fā)育等方面發(fā)揮作用[45-47],基因突變阻滯了線蟲精子的減數(shù)分裂過程[48]。在果蠅中, 去泛素化酶Usp9x被發(fā)現(xiàn)參與了性腺發(fā)育和卵子發(fā)生[49]。在甲殼動(dòng)物中, UPP被認(rèn)為通過調(diào)控蛋白質(zhì)水解速率, 在性別分化和配子發(fā)生過程中起作用[50]。在日本對(duì)蝦中, 泛素結(jié)合酶E2r ()基因在雌雄性腺中的表達(dá)呈周期性波動(dòng), 暗示著在精子發(fā)生和卵子發(fā)生過程中發(fā)揮重要作用[51]。在斑節(jié)對(duì)蝦中，通過性腺抑制性消減雜交文庫分析, 泛素連接酶E2被鑒定為性別相關(guān)基因[52]。本研究發(fā)現(xiàn)泛素化相關(guān)基因ubiquitin-activating enzyme E1、ubiquitin-conjugating enzyme E2和E3 ubiquitin-protein ligase SH3RF1以及去泛素化酶ubiquitin specific peptidase 21均在眼柄切除后的精巢中發(fā)生顯著差異表達(dá)。結(jié)果表明, 泛素/蛋白酶體途徑可能在“眼柄-促雄性腺-精巢”內(nèi)分泌軸的調(diào)控下, 在對(duì)蝦精巢的發(fā)育過程中發(fā)揮重要作用。

3.4 甲基法尼酯(methyl farnesoate, MF)代謝相關(guān)基因在性腺發(fā)育中的作用

在甲殼動(dòng)物中, 由大顎器官(mandibular organ, MO)分泌的MF起到了與昆蟲保幼激素(juvenile hormone, JH)類似的作用[53], 在甲殼動(dòng)物發(fā)育、生長(zhǎng)、蛻皮和繁殖等過程中發(fā)揮功能[54]。在昆蟲中, 保幼激素環(huán)氧化物水解酶(juvenile hormone epoxide hydrolase, JHEH)主要負(fù)責(zé)調(diào)節(jié)保幼激素JH的水平[55]。JHEH可以打開JH環(huán)氧環(huán), 生成無生物活性的JH二醇[56], 調(diào)節(jié)昆蟲的變態(tài)發(fā)育和生殖過程[57]。雖然目前沒有證據(jù)表明MF會(huì)被JHEH降解, 但在中華絨螯蟹()、日本沼蝦()和中華新米蝦()中都鑒定到基因[58], 其表達(dá)量與性成熟狀態(tài)或蛻皮周期相關(guān)[59], 推測(cè)其功能依然和MF濃度調(diào)節(jié)有關(guān)。在本研究中,在眼柄切除的精巢組織中發(fā)生顯著上調(diào)表達(dá), 暗示著其受“眼柄-促雄性腺-精巢”內(nèi)分泌軸調(diào)控參與精巢發(fā)育過程。

4 結(jié)論

本研究通過單側(cè)切除雄性對(duì)蝦的眼柄, 研究了眼柄內(nèi)分泌系統(tǒng)對(duì)胰島素樣促雄性腺素基因()和精巢內(nèi)基因表達(dá)的影響, 鑒定了精巢中多個(gè)與性別發(fā)育、內(nèi)分泌、泛素化等過程相關(guān)的差異表達(dá)基因。研究結(jié)果揭示了“眼柄-促雄性腺-精巢”內(nèi)分泌軸通過調(diào)控促雄性腺中基因及精巢中一系列基因的表達(dá)參與精巢發(fā)育的過程, 為深入理解甲殼動(dòng)物精巢發(fā)育的分子調(diào)控機(jī)制提供了重要依據(jù), 對(duì)甲殼動(dòng)物的人工繁育也具有重要指導(dǎo)意義。

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Regulatory mechanisms of the eyestalk-androgenic gland-testis endocrine axis on testis development in

CHEN Kang-xuan1, 2, LI Shi-hao1, LI Fu-hua1

(1. Key Laboratory of Experimental Marine Biology, Institute of Oceanology Chinese Academy of Sciences, Qingdao 266071, China; 2. University of Chinese Academy of Sciences, Beijing 100049, China)

The neuroendocrine system in crustacean eyestalks regulates the reproduction process. According to previous studies, the eyestalk–androgenic gland (AG)–testis endocrine axis regulates testis development in decapod crustacean species. However, the underlying molecular mechanism remains largely unknown. The Pacific white shrimp () was used in this study to investigate how unilateral eyestalk ablation regulates the expression of insulin-like AG hormones () in AG, and genes in the testis. According to the results, after unilateral eyestalk ablation, theexpression level was significantly upregulated. The transcriptome data of shrimp testis before and after unilateral eyestalk ablation were compared. A total of 267 genes were identified as differentially expressed genes (DEGS). Functional analysis showed that Some genes involved in gonad development and endocrine regulation, such as Doublesex (), juvenile hormone epoxide hydrolase (), genes related to the cytochrome P450 enzyme system, and genes related to the ubiquitylation system, were significantly upregulated. The expression of several DEGs were confirmed using quantitative real-time polymerase chain reaction (qRT-PCR), which revealed that their expression trends were consistent with those in the transcriptome data, implying that the transcriptome data were reliable. The results indicated that the endocrine system in eyestalk influenced testis development by regulating the expression ofin AG, which in turn regulated the expression of,, and genes in the endocrine and ubiquitylation systems in the testis. The results not only contribute to a better understanding of the molecular mechanisms regulating testis development but also provide significant direction for the artificial reproduction of crustaceans.

penaeid shrimp; eyestalk; insulin-like androgenic gland hormone; testis development

May 11, 2021

S917.4

A

1000-3096(2021)11-0062-11

10.11759/hykx20210511001

2021-05-11;

2021-05-19

國家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2018YFD0900202); 中以(NSFC-ISF)國際(地區(qū))合作與交流項(xiàng)目(31861143047)

[The National Key Research and Development Program of China, No. 2018YFD0900202; the Joint NSFC-ISF Research Program, No. 31861143047]

陳康軒(1996—), 男, 山東青島人, 碩士研究生, 主要從事海洋生物學(xué)研究, 電話: 13717691662, E-mail: 15561576203@163. com; 李富花(1965—),通信作者, 電話: 0532-82898836, E-mail: fhli@qdio.ac.cn

(本文編輯: 譚雪靜)