LHRH-A对尼罗罗非鱼生长及生长轴相关基因表达的影响

2013-04-19马细兰张勇周立斌刘晓春林浩然

水生生物学报 2013年1期

马细兰张勇周立斌刘晓春林浩然

(1. 中山大学有害生物控制与资源利用国家重点实验室, 生命科学学院, 水生经济动物研究所暨广东省水生经济动物良种繁育重点实验室, 广州 510275; 2. 华南师范大学生命科学学院, 广州 510631; 3. 惠州学院生命科学系, 生物技术研究所, 惠州 516007)

马细兰1,2,3张勇1周立斌3刘晓春1林浩然1

促性腺激素释放激素(GnRH)的主要作用是刺激脑垂体促性激素(GtH)的释放, 亦可促进鱼类生长激素(GH)的释放。促黄体素释放激素类似物(LHRH-A)是哺乳类GnRH的类似物, 为了分析LHRH-A对尼罗罗非鱼生长调节的作用, 设计了长期和短期2个实验, 采用腹腔注射(剂量0.1 μg/g体重)方法, 分析LHRH-A对尼罗罗非鱼绝对生长率、特定生长率、肝体系数和肥满度的影响, 并应用荧光实时定量PCR方法检测在注射LHRH-A后不同时段(6h、12h、24h、2周)尼罗罗非鱼垂体GH、肝脏GHR和肝脏IGF-I基因的表达变化。结果表明, LHRH-A组尼罗罗非鱼的绝对生长率、特定生长率、肝体系数、肥满度均显著高于对照组(P<0.05);注射LHRH-A后12h、24h垂体GH mRNA表达水平均显著升高(P<0.05), 2周后恢复到对照组水平; 注射LHRH-A后24h和2周肝脏GHR mRNA表达水平显著上升(P<0.05); 注射LHRH-A后6h肝脏IGF-I mRNA表达水平显著升高(P<0.05), 12h、24h和2周恢复到对照组水平。以上结果提示, LHRH-A可显著上调尼罗罗非鱼生长轴相关基因的表达, 从而促进鱼类的生长。

尼罗罗非鱼; 促黄体素释放激素类似物; 生长; 基因表达

促性腺激素释放激素(GnRH)的主要作用是刺激脑垂体促性激素(GtH)的释放, 已有研究表明GnRH亦可促进鲤科鱼类生长激素(Growth hormone, GH)的释放[1]。促黄体素释放激素类似物(LHRH-A)是哺乳类GnRH的类似物, 它既可刺激鲤科鱼类GH的释放又能促进鱼体生长[1]。在鲤科鱼类LHRH-A以剂量依赖方式刺激鲤鱼(Cyprinus carpio)、鲫鱼(Carassius auratus)和草鱼(Ctenopharyngodon idellus)垂体碎片基础的GtH和GH释放, 并使血清GH水平显著提高[2—5]; 在鲷科鱼类, LHRH-A可促进黑鲷(Acanthopagrus schlegeli)和黄鳍鲷(Sparu latus)的GH释放, 刺激黄鳍鲷肝脏IGF-I基因的表达, 但对黑鲷肝脏生长激素受体(Growth hormone receptor, GHR)的基因表达无影响[6,7]; 而在鲇科鱼类, LHRH-A不能促进非洲鲇鱼(Clarias gariepinus) 和野生鲇(Silurus asotus)GH的分泌和释放[8,9], 反映出LHRH-A对鱼类生长内分泌调控作用的种族特异性。尼罗罗非鱼为鲈形目丽鱼科(亦称丽鲷科)[10], 迄今未见LHRH-A对其生长调控方面的报道。

本文以尼罗罗非鱼(Oreochromis niloticus)为研究对象, 通过腹腔注射方法分析了LHRH-A的促生长作用, 并应用Real-time PCR方法检测了LHRH-A对尼罗罗非鱼生长轴相关基因(垂体GH、肝脏GHR和肝脏IGF-I)表达的影响, 旨在探讨LHRH-A对罗非鱼下丘脑-垂体-肝脏生长轴的调控作用, 以阐明LHRH-A的促生长作用机制, 为水产养殖提供理论依据。

1 材料与方法

1.1 材料

尼罗罗非鱼采自广东省番禺国家级罗非鱼良种场, 长期注射和短期注射实验的设计与处理参照文献[11]。鱼用生理盐水(PS)为实验室自配; 促黄体激素释放激素类似物(D-Ala6, Pro9-Net-LHRH, LHRHA), 购自浙江宁波激素制品厂, LHRH-A溶于PS中,剂量为0.1 μg/g体重。长期注射实验在第0、2、6、10周采样测体长、体重、肝重等, 检测LHRH-A 对生长性能的影响; 短期注射实验在第6、12、24 h采样, 用Real-time PCR方法检测LHRA-A对生长轴相关基因表达的影响。实验中所用的各种试剂及仪器设备同文献[11]。

1.2 方法

生长性能的分析选用以下几种参数指标, 计算公式如下:

式中, L1为T1时体长, L2为T2时体长, W1为T1时体重, W2为T2时体重, ΔT = T2－T1。

生长轴相关基因mRNA表达的分析用Real-time PCR检测LHRH-A 对GH、GHR、IGF-I mRNA表达的影响, 各基因的特异性引物(表1)。Real-time PCR方法参照文献[11]。

表1 荧光实时定量PCR分析所用引物Tab. 1 Primers used for real-time quantitative PCR analysis

2 结果

2.1 L HRH-A对尼罗罗非鱼生长的影响

由表2可见, 腹腔注射LHRH-A可显著促进尼罗罗非鱼生长。注射LHRH-A 2、6、10周后, 雌鱼体长、体重比对照组有显著提高(P<0.05); 6、10周后雄鱼体重显著高于对照组(P<0.05), 但体长与对照组无显著差异。与对照组相比, LHRH-A组的AGRW和AGRL都显著提高, 雌鱼和雄鱼AGRW分别提高了14.82%和15.95%, AGRL分别提高了25.36%和5.06%, 表明LHRH-A对尼罗罗非鱼促生长作用明显。LHRH-A对尼罗罗非鱼在不同发育阶段的促生长效果存在差异, 在0—2、2—6、6—10周, LHRH-A组雄鱼的SGRW分别比对照组提高了11.52%、1.21%、39.06%, 雌鱼分别提高了14.29%、6.88%、5.83%; 0—2周LHRH-A对雌鱼和雄鱼的促生长作用不存在显著差异(P>0.05), 2—6周LHRH-A对雌鱼的促生长作用显著强于对雄鱼(P<0.05), 6—10周LHRH-A对雄鱼的促生长作用极显著高于对雌鱼(P<0.01)。另外, LHRH-A显著提高了尼罗罗非鱼的肥满度(CF)和肝体系数(HSI)(P<0.05)。

2.2 LHRH-A对雄鱼GH、GHR、IGF-I mRNA表达的影响

对生长参数的影响分析中可以看出: LHRH-A可明显促进尼罗罗非鱼雄鱼和雌鱼的生长, 表明LHRH-A对雄鱼和对雌鱼生长轴相关基因表达的影响一致, 为了节省工作量, 本文只研究了LHRH-A对雄鱼生长轴相关基因(垂体GH、肝脏GHR、肝脏IGF-I)表达的影响。

对垂体GH mRNA 表达的影响注射LHRH-A 6h后, 雄鱼垂体GH mRNA表达无显著变化(P>0.05), 12h后表达量极显著提高(P<0.01), 24h后略有下降但仍显著高于对照组(P<0.05); 隔周多次注射LHRH-A, 2周后采样, 雄鱼垂体GH mRNA表达水平与对照组无显著差异(P>0.05)(表3)。

对肝脏GHR mRNA表达的影响注射LHRH-A需作用24h后才可明显提高雄鱼肝脏GHR mRNA的表达水平(P<0.05); 隔周多次注射LHRH-A, 2周后采样, 雄鱼肝脏GHR mRNA的表达仍显著高于对照组(P<0.05)(表3)。

对肝脏IGF-I mRNA表达的影响注射LHRH-A对雄鱼肝脏IGF-I mRNA表达的影响快速而短暂。注射LHRH-A 6h后, 雄鱼肝脏IGF-I mRNA表达水平显著升高(P<0.05), 12h后恢复到对照组水平, 24h后略有下降(P>0.05); 隔周多次注射LHRH-A后2周采样, LHRH-A组雄鱼肝脏IGF-I mRNA水平与对照组无显著差异(P>0.05)(表3)。

表2 LH RH-A对尼罗罗非鱼生长的影响Tab. 2 Effects of LHRH-A on the growth of O. niloticus

表3 LH RH-A对尼罗罗非鱼生长轴相关基因表达的影响Tab. 3 Effects of LHRH-A on genes expressions of the growth axis in O. niloticus

3 讨论

本研究结果表明, 腹腔注射LHRH-A可显著上调尼罗罗非鱼生长轴相关基因(垂体GH、肝脏GHR、肝脏IGF-I)的mRNA表达, 从而促进生长。

3.1 L HRH-A对垂体GH mRNA 表达的影响

研究资料显示, GnRH在离体或在体情况下都可以刺激鲤科鱼类脑垂体释放GH并促进鱼体的生长[2—5]。sGnRH和sGnRHA以剂量依赖的方式提高鲤鱼脑垂体GH mRNA水平和促进GH分泌[2], 说明GnRH不仅影响GH释放, 对GH合成也有调节作用。LHRH-A是哺乳类GnRH的高活性类似物, 能刺激鲤科鱼类和鲷科鱼类脑垂体释放GH[2—7,12,13],但不能促进非洲鲇和革胡子鲇GH的分泌和释放[8,9]。本文研究结果表明, 注射LHRH-A 6h后雄鱼垂体GH mRNA表达无显著变化(P>0.05), 12h后表达量才极显著提高(P<0.01), 24h后略有下降但仍显著高于对照组(P<0.05), 而注射LHRH-A 2周后雄鱼垂体GH mRNA表达水平与对照组无显著差异(P>0.05)。这表明LHRH-A在注射12h后可显著促进尼罗罗非鱼垂体GH mRNA表达水平, LHRH-A对丽鱼科鱼类GH的合成同样具有促进作用, 但这种促进作用在注射后2周消失, 可能是因为LHRH-A作为一种小肽, 在鱼体内不能长期保留, 2周后可能已被降解或消耗完。研究表明拌料投喂LHRH-A粗制品可显著促进鲫鱼生长而不影响其营养成分[4], 说明在生产实践中LHRH-A更适合以拌料投喂方式给予, 这样可以保证LHRH-A在体内长期起促生长作用。

3.2 L HRH-A对肝脏GHR mRNA 表达的影响

已有研究表明GnRH及其类似物对动物垂体GH的分泌和合成有明显的促进作用, 而GH对肝脏GHR的表达起重要调节作用, 由此推测GnRH及其类似物可能对动物肝脏GHR的水平及其基因表达起间接的调节作用, 有关这方面的研究资料较少。用GnRH的类似物处理大鼠后, 雄性个体肝脏GHR水平无明显改变, 雌性个体肝脏GHR水平则较对照组明显降低[14]。在鱼类中, 迄今, 只有邓利等研究过LHRH-A对黑鲷肝脏GHR的影响, 经LHRH-A刺激后, 鱼体内源性血清GH水平上升, 但肝脏GHR 基因表达水平无显著变化[6]。本研究结果表明,腹腔注射LHRH-A对尼罗罗非鱼肝脏GRH mRNA表达有显著促进作用, 这与在大鼠、黑鲷中研究结果不一致, 可能是因为所用的检测方法不同, 本研究采用了灵敏度极高的Real-time PCR检测方法, 能较准确地检测出肝脏GHR mRNA表达的差异。

3.3 L HRH-A对肝脏IGF-I mRNA 表达的影响

GH是硬骨鱼类IGF-I mRNA表达的主要调节因子之一。给性未成熟虹鳟(Oncorhynchus mykiss)注射重组大麻哈鱼GH和给银大麻哈鱼(Oncorhynchus kisutch)注射牛GH均可显著提高肝脏组织IGF-I mRNA水平[15,16]。给鲤鱼、草鱼和黑鲷注射LHRH-A亦可显著提高其血清GH 水平和明显提高其生长速率[2—7,12,13], 但有关LHRH-A对鱼类IGF-I调节的研究很少。华益民等通过体腔注射LHRH-A后发现,幼鲤和成年鲤血清GH水平升高, 同时其肝脏组织IGF-I mRNA水平也明显增高[17]。石和荣等通过投喂实验发现LHRH-A能促进黄鳍鲷GH的合成和IGF-I基因的表达[7]。本研究结果表明体腔注射LHRH-A后, 尼罗罗非鱼垂体GH mRNA的表达明显增强, 同时肝脏IGF-I mRNA 水平也显著提高,这结果与在鲤鱼和黄鳍鲷中的一致, 证明LHRH-A可通过促进垂体GH分泌和合成间接导致肝脏IGF-I mRNA表达的增强, 从而促进生长。

综上所述, LHRH-A对鱼类的促生长机理可能是: LHRH-A直接促进垂体GH分泌和合成, 从而间接引发垂体-肝脏生长轴基因表达的上调, 即肝脏GHR、IGF-I合成的增加, 表现为肝脏GHR、IGF-I mRNA表达水平的提高, 使肝脏中GHR数量增加,血液中IGF-I水平上升, 鱼类的生长加快。

[1] Lin H R. The interaction of neuroendocrine regulation on reproduction and growth in fish [J]. Zoological Research, 2000, 21(1): 12—16 [林浩然. 神经内分泌学调控鱼类生殖和生长的相互作用. 动物学研究, 2000, 21(1): 12—16]

[2] Li W S, Lin H R, Wong A O L. Effect of gonadotropin- releasing hormone (GnRH) on growth hormone (GH) secretion and gene expression in common carp pituitary [J]. Comparative Biochemistry and Physiology Part B, 2002, 132(2): 335—341

[3] Lin X W, Lin H R. In vitro studies of the effect of salmon GnRH on the growth hormone secretion by the pituitary of common carp (Cyprinus capio L.) [J]. Acta Zoologica Sinica, 1994, 40(1): 30—38 [林信伟, 林浩然. 鲑鱼促性腺素释放激素(sGnRH)调节鲤鱼脑垂体生长激素分泌的离体研究.动物学报, 1994, 40(1): 30—38]

[4] Long J, Liu X C, Xie J H, et al. Effects of Crude LHRH_A administration on growth hormone release and body growth in goldfish (Carassius auratus) [J]. Acta Scentiarum Naturalium Universitaltis SunYatseni, 2004, 43(6): 37—40 [龙进,刘晓春, 谢嘉华, 等. 投喂LHRH_A粗制品对鲫鱼生长激素释放和生长速率的影响. 中山大学学报(自然科学版), 2004, 43(6): 37—40]

[5] Sun Y, Chen L X, Lin H R. Effect of LHRH-A and 5- HT antagonist of growth hormone secretion and body growth acceleration in juvenile grass carp (Ctenopharyngodon idellus) [J]. Journal of Fishery Sciences of China, 2007, 14(3): 473—477 [孙颖, 陈练茜, 林浩然. LHRH-A和5-HT拮抗剂的协同作用对草鱼鱼种生长激素分泌活动和生长的影响. 中国水产科学, 2007, 14(3): 473—477]

[6] Deng L, Lin H R. Effects of Luteinizing Hormone-releasing hormone analogue injection on growth hormone and its receptor in black seabream [J]. Journal of Shenzhen University (Science & Engineering), 2003, 20(12): 60—65 [邓利, 林浩然. 腹腔注射LHRH-A对黑鲷生长激素及其受体的影响.深圳大学学报(理工版), 2003, 20(12): 60—65]

[7] Shi H R, Zhang Y, Zhang W M, et al. Effect of cysteamine hydrochloride and luteininzing hormone-releasing hormone analog on the growth and the expression of IGF-ⅠmRNA in the yellow-fin porgy Sparus latus [J]. Acta Zoologica Sinica, 2005, 51(1): 108—116 [石和荣, 张勇, 张为民, 等. 半胱胺盐酸盐和LHRH-A对黄鳍鲷IGF-Ⅰ基因表达和生长的影响.动物学报, 2005, 51(1): 108—116]

[8] Bosma P T, Van Dijk W, Van Haren S, et al. GnRH receptors are restricted to gonadotropes in male African catfish [C]. In:Goetz F W, P Thomas (Eds.), Proceedings of the 5thInternational Sympoium on Reproductive Physiology of Fish. Austin, Texas, U.S.A. 1995, 2—8

[9] Wen H S, Lin H R, Xiao D, et al. Seasonal changes and neuroendocrine regulaion of growth hormone secretion in feral catfish (Silurus asotus) [J]. Acta Zoologica Sinica, 2002, 48(2): 213—220 [温海深, 林浩然, 肖东, 等. 野生鲇鱼生长激素分泌的季节变化及其神经内分泌调控. 动物学报, 2002, 48(2): 213—220]

[10] Lin X, Lin S G, Wang Q X, et al. Study on the histochemical property of the mast cells in the digestive tract of nile tilapia [J]. Acta Hydrobiologica Sinica, 2011, 35(1): 132—137 [林旋, 林树根, 王全溪, 等. 尼罗罗非鱼消化道肥大细胞的组化性质. 水生生物学报, 2011, 35(1): 132—137]

[11] Ma X L, Zhang Y, Liu X C, et al. Effects of CSH on the growth and gene expressions of the growth axis in Nile tilapia Orechromis niloticus [J] . Oceanologia et Limnologia Sinica, 2010, 41(2): 240—245[马细兰, 张勇, 刘晓春, 等.半胱胺盐酸盐(CSH)对尼罗罗非鱼(Oreochromis niloticus)生长及生长轴相关基因表达的影响. 海洋与湖沼, 2010, 41(2): 240—245]

[12] Wang L, Lin H R. Effects of LHRH-A and DA on growth hormone secretion in juvenile and mature female common carp (Cyprinus carpio) [J]. Acta Zoologica Sinica, 1997, 43(3): 303—308 [王黎, 林浩然. 促黄体素释放激素类似物和多巴胺对鲤鱼幼鱼和性成熟雌鱼生长激素分泌的影响. 动物学报, 1997, 43(3): 303—308]

[13] Lin H R, LU M, Lin X W. Effects of gonadotropin releasing hormone(GnRH) analogs and sex steroids on growth hormone (GH) secretion and growth in common carp (Cyprinus carpio) and grass carp (Ctenopharyngodon idellus) [J]. Aquaculture, 1995, 135(2): 173—184

[14] Gevers E F, Wit J M, Robinson I C A. Effects of long-term gonadtropin-releasing hormone analog treatment on growth, growth hormone (GH) secretion, GH receptors, and GH-binding protein in the rat [J]. Pediatric Research, 1998, 43(1): 111—120

[15] Duan C, Hirano T. Hormonal regulation of insulin-like growth factor (IGF-I) mRNA expression in coho salmon [J]. American Zoologist, 1992, 32(1): 32—36

[16] Cao Q P, Duguay S J, Plisetskaya E, et al. Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factor I mRNA [J]. Molecular Endocrinology, 1989, 3(12): 2005—2010

[17] Hua Y M, Lin H R. Differential expression and effect of LHRH-A on expression of IGF-I mRNA in juvenile and adult common carp (Cyprinus carpio) [J]. Acta Hydrobiologica Sinica, 2001, 25(5): 498—502 [华益民, 林浩然. IGF-I mRNA 在不同年龄鲤表达的差异和LHRH-A对IGF-I mRNA 表达的影响. 水生生物学报, 2001, 25(5): 498—502]

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

1000-3207(2013)01-0042-06

10.7541/2013.42

2011-10-20;

2012-09-17

国家重点基础研究发展计划(973计划)项目(2010CB126302); 广东省自然科学基金项目(9451601501001986); 广东省教育部产学研结合项目(2010B090400551)资助

马细兰(1976—), 女, 广东南雄人; 博士, 副教授; 主要从事鱼类生理及分子生物学研究。E-mail: mxl@hzu.edu.cn

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