馬細(xì)蘭張 勇周立斌劉曉春林浩然

(1. 中山大學(xué)有害生物控制與資源利用國家重點(diǎn)實(shí)驗(yàn)室, 生命科學(xué)學(xué)院, 水生經(jīng)濟(jì)動(dòng)物研究所暨廣東省水生經(jīng)濟(jì)動(dòng)物良種繁育重點(diǎn)實(shí)驗(yàn)室, 廣州 510275; 2. 華南師范大學(xué)生命科學(xué)學(xué)院, 廣州 510631; 3. 惠州學(xué)院生命科學(xué)系, 生物技術(shù)研究所, 惠州 516007)

LHRH-A對(duì)尼羅羅非魚生長及生長軸相關(guān)基因表達(dá)的影響

馬細(xì)蘭1,2,3張 勇1周立斌3劉曉春1林浩然1

(1. 中山大學(xué)有害生物控制與資源利用國家重點(diǎn)實(shí)驗(yàn)室, 生命科學(xué)學(xué)院, 水生經(jīng)濟(jì)動(dòng)物研究所暨廣東省水生經(jīng)濟(jì)動(dòng)物良種繁育重點(diǎn)實(shí)驗(yàn)室, 廣州 510275; 2. 華南師范大學(xué)生命科學(xué)學(xué)院, 廣州 510631; 3. 惠州學(xué)院生命科學(xué)系, 生物技術(shù)研究所, 惠州 516007)

促性腺激素釋放激素(GnRH)的主要作用是刺激腦垂體促性激素(GtH)的釋放, 亦可促進(jìn)魚類生長激素(GH)的釋放。促黃體素釋放激素類似物(LHRH-A)是哺乳類GnRH的類似物, 為了分析LHRH-A對(duì)尼羅羅非魚生長調(diào)節(jié)的作用, 設(shè)計(jì)了長期和短期2個(gè)實(shí)驗(yàn), 采用腹腔注射(劑量0.1 μg/g體重)方法, 分析LHRH-A對(duì)尼羅羅非魚絕對(duì)生長率、特定生長率、肝體系數(shù)和肥滿度的影響, 并應(yīng)用熒光實(shí)時(shí)定量PCR方法檢測在注射LHRH-A后不同時(shí)段(6h、12h、24h、2周)尼羅羅非魚垂體GH、肝臟GHR和肝臟IGF-I基因的表達(dá)變化。結(jié)果表明, LHRH-A組尼羅羅非魚的絕對(duì)生長率、特定生長率、肝體系數(shù)、肥滿度均顯著高于對(duì)照組(P<0.05);注射LHRH-A后12h、24h垂體GH mRNA表達(dá)水平均顯著升高(P<0.05), 2周后恢復(fù)到對(duì)照組水平; 注射LHRH-A后24h和2周肝臟GHR mRNA表達(dá)水平顯著上升(P<0.05); 注射LHRH-A后6h肝臟IGF-I mRNA表達(dá)水平顯著升高(P<0.05), 12h、24h和2周恢復(fù)到對(duì)照組水平。以上結(jié)果提示, LHRH-A可顯著上調(diào)尼羅羅非魚生長軸相關(guān)基因的表達(dá), 從而促進(jìn)魚類的生長。

尼羅羅非魚; 促黃體素釋放激素類似物; 生長; 基因表達(dá)

促性腺激素釋放激素(GnRH)的主要作用是刺激腦垂體促性激素(GtH)的釋放, 已有研究表明GnRH亦可促進(jìn)鯉科魚類生長激素(Growth hormone, GH)的釋放[1]。促黃體素釋放激素類似物(LHRH-A)是哺乳類GnRH的類似物, 它既可刺激鯉科魚類GH的釋放又能促進(jìn)魚體生長[1]。在鯉科魚類LHRH-A以劑量依賴方式刺激鯉魚(Cyprinus carpio)、鯽魚(Carassius auratus)和草魚(Ctenopharyngodon idellus)垂體碎片基礎(chǔ)的GtH和GH釋放, 并使血清GH水平顯著提高[2—5]; 在鯛科魚類, LHRH-A可促進(jìn)黑鯛(Acanthopagrus schlegeli)和黃鰭鯛(Sparu latus)的GH釋放, 刺激黃鰭鯛肝臟IGF-I基因的表達(dá), 但對(duì)黑鯛肝臟生長激素受體(Growth hormone receptor, GHR)的基因表達(dá)無影響[6,7]; 而在鲇科魚類, LHRH-A不能促進(jìn)非洲鲇魚(Clarias gariepinus) 和野生鲇(Silurus asotus)GH的分泌和釋放[8,9], 反映出LHRH-A對(duì)魚類生長內(nèi)分泌調(diào)控作用的種族特異性。尼羅羅非魚為鱸形目麗魚科(亦稱麗鯛科)[10], 迄今未見LHRH-A對(duì)其生長調(diào)控方面的報(bào)道。

本文以尼羅羅非魚(Oreochromis niloticus)為研究對(duì)象, 通過腹腔注射方法分析了LHRH-A的促生長作用, 并應(yīng)用Real-time PCR方法檢測了LHRH-A對(duì)尼羅羅非魚生長軸相關(guān)基因(垂體GH、肝臟GHR和肝臟IGF-I)表達(dá)的影響, 旨在探討LHRH-A對(duì)羅非魚下丘腦-垂體-肝臟生長軸的調(diào)控作用, 以闡明LHRH-A的促生長作用機(jī)制, 為水產(chǎn)養(yǎng)殖提供理論依據(jù)。

1 材料與方法

1.1 材料

尼羅羅非魚采自廣東省番禺國家級(jí)羅非魚良種場, 長期注射和短期注射實(shí)驗(yàn)的設(shè)計(jì)與處理參照文獻(xiàn)[11]。魚用生理鹽水(PS)為實(shí)驗(yàn)室自配; 促黃體激素釋放激素類似物(D-Ala6, Pro9-Net-LHRH, LHRHA), 購自浙江寧波激素制品廠, LHRH-A溶于PS中,劑量為0.1 μg/g體重。長期注射實(shí)驗(yàn)在第0、2、6、10周采樣測體長、體重、肝重等, 檢測LHRH-A 對(duì)生長性能的影響; 短期注射實(shí)驗(yàn)在第6、12、24 h采樣, 用Real-time PCR方法檢測LHRA-A對(duì)生長軸相關(guān)基因表達(dá)的影響。實(shí)驗(yàn)中所用的各種試劑及儀器設(shè)備同文獻(xiàn)[11]。

1.2 方法

生長性能的分析 選用以下幾種參數(shù)指標(biāo), 計(jì)算公式如下:

式中, L1為T1時(shí)體長, L2為T2時(shí)體長, W1為T1時(shí)體重, W2為T2時(shí)體重, ΔT = T2－T1。

生長軸相關(guān)基因mRNA表達(dá)的分析 用Real-time PCR檢測LHRH-A 對(duì)GH、GHR、IGF-I mRNA表達(dá)的影響, 各基因的特異性引物(表1)。Real-time PCR方法參照文獻(xiàn)[11]。

表1 熒光實(shí)時(shí)定量PCR分析所用引物Tab. 1 Primers used for real-time quantitative PCR analysis

2 結(jié)果

2.1 L HRH-A對(duì)尼羅羅非魚生長的影響

由表2可見, 腹腔注射LHRH-A可顯著促進(jìn)尼羅羅非魚生長。注射LHRH-A 2、6、10周后, 雌魚體長、體重比對(duì)照組有顯著提高(P<0.05); 6、10周后雄魚體重顯著高于對(duì)照組(P<0.05), 但體長與對(duì)照組無顯著差異。與對(duì)照組相比, LHRH-A組的AGRW和AGRL都顯著提高, 雌魚和雄魚AGRW分別提高了14.82%和15.95%, AGRL分別提高了25.36%和5.06%, 表明LHRH-A對(duì)尼羅羅非魚促生長作用明顯。LHRH-A對(duì)尼羅羅非魚在不同發(fā)育階段的促生長效果存在差異, 在0—2、2—6、6—10周, LHRH-A組雄魚的SGRW分別比對(duì)照組提高了11.52%、1.21%、39.06%, 雌魚分別提高了14.29%、6.88%、5.83%; 0—2周LHRH-A對(duì)雌魚和雄魚的促生長作用不存在顯著差異(P>0.05), 2—6周LHRH-A對(duì)雌魚的促生長作用顯著強(qiáng)于對(duì)雄魚(P<0.05), 6—10周LHRH-A對(duì)雄魚的促生長作用極顯著高于對(duì)雌魚(P<0.01)。另外, LHRH-A顯著提高了尼羅羅非魚的肥滿度(CF)和肝體系數(shù)(HSI)(P<0.05)。

2.2 LHRH-A對(duì)雄魚GH、GHR、IGF-I mRNA表達(dá)的影響

對(duì)生長參數(shù)的影響分析中可以看出: LHRH-A可明顯促進(jìn)尼羅羅非魚雄魚和雌魚的生長, 表明LHRH-A對(duì)雄魚和對(duì)雌魚生長軸相關(guān)基因表達(dá)的影響一致, 為了節(jié)省工作量, 本文只研究了LHRH-A對(duì)雄魚生長軸相關(guān)基因(垂體GH、肝臟GHR、肝臟IGF-I)表達(dá)的影響。

對(duì)垂體GH mRNA 表達(dá)的影響 注射LHRH-A 6h后, 雄魚垂體GH mRNA表達(dá)無顯著變化(P>0.05), 12h后表達(dá)量極顯著提高(P<0.01), 24h后略有下降但仍顯著高于對(duì)照組(P<0.05); 隔周多次注射LHRH-A, 2周后采樣, 雄魚垂體GH mRNA表達(dá)水平與對(duì)照組無顯著差異(P>0.05)(表3)。

對(duì)肝臟GHR mRNA表達(dá)的影響 注射LHRH-A需作用24h后才可明顯提高雄魚肝臟GHR mRNA的表達(dá)水平(P<0.05); 隔周多次注射LHRH-A, 2周后采樣, 雄魚肝臟GHR mRNA的表達(dá)仍顯著高于對(duì)照組(P<0.05)(表3)。

對(duì)肝臟IGF-I mRNA表達(dá)的影響 注射LHRH-A對(duì)雄魚肝臟IGF-I mRNA表達(dá)的影響快速而短暫。注射LHRH-A 6h后, 雄魚肝臟IGF-I mRNA表達(dá)水平顯著升高(P<0.05), 12h后恢復(fù)到對(duì)照組水平, 24h后略有下降(P>0.05); 隔周多次注射LHRH-A后2周采樣, LHRH-A組雄魚肝臟IGF-I mRNA水平與對(duì)照組無顯著差異(P>0.05)(表3)。

表2 LH RH-A對(duì)尼羅羅非魚生長的影響Tab. 2 Effects of LHRH-A on the growth of O. niloticus

表3 LH RH-A對(duì)尼羅羅非魚生長軸相關(guān)基因表達(dá)的影響Tab. 3 Effects of LHRH-A on genes expressions of the growth axis in O. niloticus

3 討論

本研究結(jié)果表明, 腹腔注射LHRH-A可顯著上調(diào)尼羅羅非魚生長軸相關(guān)基因(垂體GH、肝臟GHR、肝臟IGF-I)的mRNA表達(dá), 從而促進(jìn)生長。

3.1 L HRH-A對(duì)垂體GH mRNA 表達(dá)的影響

研究資料顯示, GnRH在離體或在體情況下都可以刺激鯉科魚類腦垂體釋放GH并促進(jìn)魚體的生長[2—5]。sGnRH和sGnRHA以劑量依賴的方式提高鯉魚腦垂體GH mRNA水平和促進(jìn)GH分泌[2], 說明GnRH不僅影響GH釋放, 對(duì)GH合成也有調(diào)節(jié)作用。LHRH-A是哺乳類GnRH的高活性類似物, 能刺激鯉科魚類和鯛科魚類腦垂體釋放GH[2—7,12,13],但不能促進(jìn)非洲鲇和革胡子鲇GH的分泌和釋放[8,9]。本文研究結(jié)果表明, 注射LHRH-A 6h后雄魚垂體GH mRNA表達(dá)無顯著變化(P>0.05), 12h后表達(dá)量才極顯著提高(P<0.01), 24h后略有下降但仍顯著高于對(duì)照組(P<0.05), 而注射LHRH-A 2周后雄魚垂體GH mRNA表達(dá)水平與對(duì)照組無顯著差異(P>0.05)。這表明LHRH-A在注射12h后可顯著促進(jìn)尼羅羅非魚垂體GH mRNA表達(dá)水平, LHRH-A對(duì)麗魚科魚類GH的合成同樣具有促進(jìn)作用, 但這種促進(jìn)作用在注射后2周消失, 可能是因?yàn)長HRH-A作為一種小肽, 在魚體內(nèi)不能長期保留, 2周后可能已被降解或消耗完。研究表明拌料投喂LHRH-A粗制品可顯著促進(jìn)鯽魚生長而不影響其營養(yǎng)成分[4], 說明在生產(chǎn)實(shí)踐中LHRH-A更適合以拌料投喂方式給予, 這樣可以保證LHRH-A在體內(nèi)長期起促生長作用。

3.2 L HRH-A對(duì)肝臟GHR mRNA 表達(dá)的影響

已有研究表明GnRH及其類似物對(duì)動(dòng)物垂體GH的分泌和合成有明顯的促進(jìn)作用, 而GH對(duì)肝臟GHR的表達(dá)起重要調(diào)節(jié)作用, 由此推測GnRH及其類似物可能對(duì)動(dòng)物肝臟GHR的水平及其基因表達(dá)起間接的調(diào)節(jié)作用, 有關(guān)這方面的研究資料較少。用GnRH的類似物處理大鼠后, 雄性個(gè)體肝臟GHR水平無明顯改變, 雌性個(gè)體肝臟GHR水平則較對(duì)照組明顯降低[14]。在魚類中, 迄今, 只有鄧?yán)妊芯窟^LHRH-A對(duì)黑鯛肝臟GHR的影響, 經(jīng)LHRH-A刺激后, 魚體內(nèi)源性血清GH水平上升, 但肝臟GHR 基因表達(dá)水平無顯著變化[6]。本研究結(jié)果表明,腹腔注射LHRH-A對(duì)尼羅羅非魚肝臟GRH mRNA表達(dá)有顯著促進(jìn)作用, 這與在大鼠、黑鯛中研究結(jié)果不一致, 可能是因?yàn)樗玫臋z測方法不同, 本研究采用了靈敏度極高的Real-time PCR檢測方法, 能較準(zhǔn)確地檢測出肝臟GHR mRNA表達(dá)的差異。

3.3 L HRH-A對(duì)肝臟IGF-I mRNA 表達(dá)的影響

GH是硬骨魚類IGF-I mRNA表達(dá)的主要調(diào)節(jié)因子之一。給性未成熟虹鱒(Oncorhynchus mykiss)注射重組大麻哈魚GH和給銀大麻哈魚(Oncorhynchus kisutch)注射牛GH均可顯著提高肝臟組織IGF-I mRNA水平[15,16]。給鯉魚、草魚和黑鯛注射LHRH-A亦可顯著提高其血清GH 水平和明顯提高其生長速率[2—7,12,13], 但有關(guān)LHRH-A對(duì)魚類IGF-I調(diào)節(jié)的研究很少。華益民等通過體腔注射LHRH-A后發(fā)現(xiàn),幼鯉和成年鯉血清GH水平升高, 同時(shí)其肝臟組織IGF-I mRNA水平也明顯增高[17]。石和榮等通過投喂實(shí)驗(yàn)發(fā)現(xiàn)LHRH-A能促進(jìn)黃鰭鯛GH的合成和IGF-I基因的表達(dá)[7]。本研究結(jié)果表明體腔注射LHRH-A后, 尼羅羅非魚垂體GH mRNA的表達(dá)明顯增強(qiáng), 同時(shí)肝臟IGF-I mRNA 水平也顯著提高,這結(jié)果與在鯉魚和黃鰭鯛中的一致, 證明LHRH-A可通過促進(jìn)垂體GH分泌和合成間接導(dǎo)致肝臟IGF-I mRNA表達(dá)的增強(qiáng), 從而促進(jìn)生長。

綜上所述, LHRH-A對(duì)魚類的促生長機(jī)理可能是: LHRH-A直接促進(jìn)垂體GH分泌和合成, 從而間接引發(fā)垂體-肝臟生長軸基因表達(dá)的上調(diào), 即肝臟GHR、IGF-I合成的增加, 表現(xiàn)為肝臟GHR、IGF-I mRNA表達(dá)水平的提高, 使肝臟中GHR數(shù)量增加,血液中IGF-I水平上升, 魚類的生長加快。

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EFFECTS OF LHRH-A ON THE GROWTH AND GENE EXPRESSIONS OF THE GROWTH AXIS IN NILE TILAPIA OREOCHROMIS NILOTICUS

MA Xi-Lan1,2,3, ZHANG Yong1, ZHOU Li-Bin3, LIU Xiao-Chun1and LIN Hao-Ran1
(1. State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China; 2. School of Life Sciences, South China Normal University, Guangzhou 510631, China; 3. Department of Life Science, Huizhou University and Institute of Biotechnology, Huizhou 516007, China)

Regulations of synthesis and secretion of growth hormone (GH) in teleosts are based on the dual control of hypothalamic stimulators including gonadotropin-releasing hormone (GnRH), dopamine (DA), and growth hormone-releasing hormone (GHRH), and hypothalamic inhibitors including somatostatin (SS) and norepinephrine (NE). Gonadotropin-releasing hormone (GnRH) can stimulate the release of growth hormone (GH). LHRH-A is an analog of mammalian GnRH. Nile tilapia (Oreochromis niloticus), a freshwater fish with sexual dimorphism. The effects of LHRH-A on the growth and expressions of GH, GHR and IGF-I mRNA in Nile tilapia remain unclear. Two experiments, designated as long-term and short-term experiments, were carried out. In the long-term experiment, two hundreds Nile tilapias of similar body weight were randomly assigned to the control group (intraperitoneal injection with phosphate saline) and the LHRH-A group (intraperitoneal injection with LHRH-A, 0.01 μg/g body weight) and fed under the same conditions for 70 days. The absolute growth rates (AGR), specific growth rates (SGR), condition factor (CF) and hepatosomatic index (HSI) were obtained by measuring the body length, body weight and hepatic weight at different stages. AGRWand AGRLlevels of LHRH-A-treated male and female increased by 15.95%, 14.82% and 5.06%, 25.36%, respectively, compared to control group (P<0.05). During the 0—2, 2—6, and 6—10 weeks, SGRWand AGRLlevels of LHRH-A-treated male increased by 11.52%, 1.21%, 39.06% and 3.94%, 1.08%, 10.00%, respectively, compared to the control group (P<0.05); similarly, SGRWand AGRLlevels of LHRH-A-treated female increased by 14.29%, 6.88%, 5.83% and 24.19%, 9.09%, 28.21%, respectively (P<0.05). Furthermore, CF and HIS levels of the LHRH-A group were also significantly higher than those of the control group (P<0.05). In the short-term experiment, 120 Nile tilapias of similar body weight were randomly assigned to the control group (intraperitoneal injection with phosphate saline) and the LHRH-A group (intraperitoneal injection with LHRH-A, 0.01 μg/g body weight) and sampled at 6, 12, 24h, and 2 week. Real-time quantitative PCR was used to detect the expressions of GH in pituitary and GHR and IGF-I in liver at different life stages after injection. The expressions of GH in pituitary increased at 12h (P<0.05), and IGF-I in liver increased quickly at 6h, but soon returned to the level of control group. The expression of GHR in liver increased slowly at 24h (P<0.05) and maintained high at 2 week (P<0.01). The results indicated that LHRH-A could increase the mRNA expressions of GH in pituitary and GHR and IGF-I in liver, and significantly promote growth of Nile tilapia. Moreover, our study suggested that large scale administration of some neruoendocrine factor in diet to accelerate the growth rate of cultured fish has promising potential.

Nile tilapia Oreochromis niloticus; LHRH-A; Growth; Gene expression

Q344

A

1000-3207(2013)01-0042-06

10.7541/2013.42

2011-10-20;

2012-09-17

國家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973計(jì)劃)項(xiàng)目(2010CB126302); 廣東省自然科學(xué)基金項(xiàng)目(9451601501001986); 廣東省教育部產(chǎn)學(xué)研結(jié)合項(xiàng)目(2010B090400551)資助

馬細(xì)蘭(1976—), 女, 廣東南雄人; 博士, 副教授; 主要從事魚類生理及分子生物學(xué)研究。E-mail: mxl@hzu.edu.cn

林浩然(1934—), 男, 中國工程院院士; E-mail：lsslhr@mail.sysu.edu.cn