LHRH-A對尼羅羅非魚生長及生長軸相關基因表達的影響

馬細蘭張 勇周立斌劉曉春林浩然

(1. 中山大學有害生物控制與資源利用國家重點實驗室, 生命科學學院, 水生經(jīng)濟動物研究所暨廣東省水生經(jīng)濟動物良種繁育重點實驗室, 廣州 510275; 2. 華南師范大學生命科學學院, 廣州 510631; 3. 惠州學院生命科學系, 生物技術研究所, 惠州 516007)

LHRH-A對尼羅羅非魚生長及生長軸相關基因表達的影響

馬細蘭1,2,3張 勇1周立斌3劉曉春1林浩然1

(1. 中山大學有害生物控制與資源利用國家重點實驗室, 生命科學學院, 水生經(jīng)濟動物研究所暨廣東省水生經(jīng)濟動物良種繁育重點實驗室, 廣州 510275; 2. 華南師范大學生命科學學院, 廣州 510631; 3. 惠州學院生命科學系, 生物技術研究所, 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對尼羅羅非魚生長調節(jié)的作用, 設計了長期和短期2個實驗, 采用腹腔注射(劑量0.1 μg/g體重)方法, 分析LHRH-A對尼羅羅非魚絕對生長率、特定生長率、肝體系數(shù)和肥滿度的影響, 并應用熒光實時定量PCR方法檢測在注射LHRH-A后不同時段(6h、12h、24h、2周)尼羅羅非魚垂體GH、肝臟GHR和肝臟IGF-I基因的表達變化。結果表明, LHRH-A組尼羅羅非魚的絕對生長率、特定生長率、肝體系數(shù)、肥滿度均顯著高于對照組(P<0.05);注射LHRH-A后12h、24h垂體GH mRNA表達水平均顯著升高(P<0.05), 2周后恢復到對照組水平; 注射LHRH-A后24h和2周肝臟GHR mRNA表達水平顯著上升(P<0.05); 注射LHRH-A后6h肝臟IGF-I mRNA表達水平顯著升高(P<0.05), 12h、24h和2周恢復到對照組水平。以上結果提示, LHRH-A可顯著上調尼羅羅非魚生長軸相關基因的表達, 從而促進魚類的生長。

尼羅羅非魚; 促黃體素釋放激素類似物; 生長; 基因表達

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, 但對黑鯛肝臟生長激素受體(Growth hormone receptor, GHR)的基因表達無影響[6,7]; 而在鲇科魚類, LHRH-A不能促進非洲鲇魚(Clarias gariepinus) 和野生鲇(Silurus asotus)GH的分泌和釋放[8,9], 反映出LHRH-A對魚類生長內分泌調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對其生長調控方面的報道。

本文以尼羅羅非魚(Oreochromis niloticus)為研究對象, 通過腹腔注射方法分析了LHRH-A的促生長作用, 并應用Real-time PCR方法檢測了LHRH-A對尼羅羅非魚生長軸相關基因(垂體GH、肝臟GHR和肝臟IGF-I)表達的影響, 旨在探討LHRH-A對羅非魚下丘腦-垂體-肝臟生長軸的調控作用, 以闡明LHRH-A的促生長作用機制, 為水產養(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羅非魚良種場, 長期注射和短期注射實驗的設計與處理參照文獻[11]。魚用生理鹽水(PS)為實驗室自配; 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。長期注射實驗在第0、2、6、10周采樣測體長、體重、肝重等, 檢測LHRH-A 對生長性能的影響; 短期注射實驗在第6、12、24 h采樣, 用Real-time PCR方法檢測LHRA-A對生長軸相關基因表達的影響。實驗中所用的各種試劑及儀器設備同文獻[11]。

1.2 方法

生長性能的分析 選用以下幾種參數(shù)指標, 計算公式如下:

式中, L1為T1時體長, L2為T2時體長, W1為T1時體重, W2為T2時體重, ΔT = T2－T1。

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

表1 熒光實時定量PCR分析所用引物Tab. 1 Primers used for real-time quantitative PCR analysis

2 結果

2.1 L HRH-A對尼羅羅非魚生長的影響

由表2可見, 腹腔注射LHRH-A可顯著促進尼羅羅非魚生長。注射LHRH-A 2、6、10周后, 雌魚體長、體重比對照組有顯著提高(P<0.05); 6、10周后雄魚體重顯著高于對照組(P<0.05), 但體長與對照組無顯著差異。與對照組相比, 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對尼羅羅非魚促生長作用明顯。LHRH-A對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對雌魚的促生長作用顯著強于對雄魚(P<0.05), 6—10周LHRH-A對雄魚的促生長作用極顯著高于對雌魚(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對雄魚GH、GHR、IGF-I mRNA表達的影響

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

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

對肝臟GHR mRNA表達的影響 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的表達仍顯著高于對照組(P<0.05)(表3)。

對肝臟IGF-I mRNA表達的影響 注射LHRH-A對雄魚肝臟IGF-I mRNA表達的影響快速而短暫。注射LHRH-A 6h后, 雄魚肝臟IGF-I mRNA表達水平顯著升高(P<0.05), 12h后恢復到對照組水平, 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水平與對照組無顯著差異(P>0.05)(表3)。

表2 LH RH-A對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對尼羅羅非魚生長軸相關基因表達的影響Tab. 3 Effects of LHRH-A on genes expressions of the growth axis in O. niloticus

3 討論

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

3.1 L HRH-A對垂體GH mRNA 表達的影響

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GH分泌[2], 說明GnRH不僅影響GH釋放, 對GH合成也有調節(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],但不能促進非洲鲇和革胡子鲇GH的分泌和釋放[8,9]。本文研究結果表明, 注射LHRH-A 6h后雄魚垂體GH mRNA表達無顯著變化(P>0.05), 12h后表達量才極顯著提高(P<0.01), 24h后略有下降但仍顯著高于對照組(P<0.05), 而注射LHRH-A 2周后雄魚垂體GH mRNA表達水平與對照組無顯著差異(P>0.05)。這表明LHRH-A在注射12h后可顯著促進尼羅羅非魚垂體GH mRNA表達水平, LHRH-A對麗魚科魚類GH的合成同樣具有促進作用, 但這種促進作用在注射后2周消失, 可能是因為LHRH-A作為一種小肽, 在魚體內不能長期保留, 2周后可能已被降解或消耗完。研究表明拌料投喂LHRH-A粗制品可顯著促進鯽魚生長而不影響其營養(yǎng)成分[4], 說明在生產實踐中LHRH-A更適合以拌料投喂方式給予, 這樣可以保證LHRH-A在體內長期起促生長作用。

3.2 L HRH-A對肝臟GHR mRNA 表達的影響

已有研究表明GnRH及其類似物對動物垂體GH的分泌和合成有明顯的促進作用, 而GH對肝臟GHR的表達起重要調節(jié)作用, 由此推測GnRH及其類似物可能對動物肝臟GHR的水平及其基因表達起間接的調節(jié)作用, 有關這方面的研究資料較少。用GnRH的類似物處理大鼠后, 雄性個體肝臟GHR水平無明顯改變, 雌性個體肝臟GHR水平則較對照組明顯降低[14]。在魚類中, 迄今, 只有鄧利等研究過LHRH-A對黑鯛肝臟GHR的影響, 經(jīng)LHRH-A刺激后, 魚體內源性血清GH水平上升, 但肝臟GHR 基因表達水平無顯著變化[6]。本研究結果表明,腹腔注射LHRH-A對尼羅羅非魚肝臟GRH mRNA表達有顯著促進作用, 這與在大鼠、黑鯛中研究結果不一致, 可能是因為所用的檢測方法不同, 本研究采用了靈敏度極高的Real-time PCR檢測方法, 能較準確地檢測出肝臟GHR mRNA表達的差異。

3.3 L HRH-A對肝臟IGF-I mRNA 表達的影響

GH是硬骨魚類IGF-I mRNA表達的主要調節(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LHRH-A對魚類IGF-I調節(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驗發(fā)現(xiàn)LHRH-A能促進黃鰭鯛GH的合成和IGF-I基因的表達[7]。本研究結果表明體腔注射LHRH-A后, 尼羅羅非魚垂體GH mRNA的表達明顯增強, 同時肝臟IGF-I mRNA 水平也顯著提高,這結果與在鯉魚和黃鰭鯛中的一致, 證明LHRH-A可通過促進垂體GH分泌和合成間接導致肝臟IGF-I mRNA表達的增強, 從而促進生長。

綜上所述, LHRH-A對魚類的促生長機理可能是: LHRH-A直接促進垂體GH分泌和合成, 從而間接引發(fā)垂體-肝臟生長軸基因表達的上調, 即肝臟GHR、IGF-I合成的增加, 表現(xiàn)為肝臟GHR、IGF-I mRNA表達水平的提高, 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

國家重點基礎研究發(fā)展計劃(973計劃)項目(2010CB126302); 廣東省自然科學基金項目(9451601501001986); 廣東省教育部產學研結合項目(2010B090400551)資助

馬細蘭(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