畜牧兽医学报  2018, Vol. 49 Issue (2): 263-269. DOI: 10.11843/j.issn.0366-6964.2018.02.005    PDF    
EYA 3和TSH β在季节性发情和常年发情绵羊中的表达模式
夏青1, 张效生2, 刘秋月1, 王翔宇1, 贺小云1, 郭晓飞1, 胡文萍1, 张金龙2, 储明星1, 狄冉1     
1. 中国农业科学院北京畜牧兽医研究所, 农业部动物遗传育种与繁殖重点实验室, 北京 100193;
2. 天津市畜牧兽医研究所, 天津 300381
摘要:本研究旨在揭示EYA 3和TSH β两个基因在绵羊季节性发情调控和繁殖时期转换中的作用。采用q-PCR技术分析眼缺失基因3(Eyes absent 3,EYA 3)与促甲状腺激素β亚基基因(Thyrotrophin β subunit,TSH β)在不同光照条件下(长光照和短光照)季节性发情的苏尼特羊(Sunite sheep,SNT)和不同繁殖时期(黄体期和卵泡期)常年发情的小尾寒羊(Small Tail Han sheep,STH)各组织中的表达模式。结果表明,EYA 3基因在两个品种绵羊各组织中广泛表达,TSH β基因仅在绵羊垂体中高表达;在绝大多数组织中,两基因在苏尼特羊中表达量为长光照(Long photoperiod,LP)高于短光照(Short photoperiod,SP),而在小尾寒羊中是黄体期高于卵泡期;SP转至LP时,苏尼特羊垂体中EYA 3与TSH β的表达量均升高,在长光照第3天(Long photoperiod 3,LP 3)EYA 3表达量达到最高,在LP 21时,TSH β表达量达到最高,EYA 3表达量提前于TSH β开始降低。本研究揭示了EYA 3与TSH β在常年发情和季节性发情绵羊中的表达谱特征,不同光照条件与不同繁殖时期绵羊松果体与垂体中上述基因的表达模式,暗示EYA 3可能也在松果体部位发挥作用,并对绵羊季节性发情进行调控,EYA 3与TSH β两个基因也可能参与发情时期的转换;SP转变为LP过程中,EYA 3早于TSH β发挥调控作用。
关键词绵羊    EYA 3    TSH β    季节性发情    繁殖时期    表达模式    
Expression Patterns of EYA 3 and TSH β in Seasonal Estrous and Year-round Estrous Sheep
XIA Qing1, ZHANG Xiao-sheng2, LIU Qiu-yue1, WANG Xiang-yu1, HE Xiao-yun1, GUO Xiao-fei1, HU Wen-ping1, ZHANG Jin-long2, CHU Ming-xing1, DI Ran1     
1. Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. Tianjin Institute of Animal Science, Tianjin 300381, China
Abstract: The aim of this study was to reveal EYA 3 and TSH β regulatory roles in seasonal estrus and reproduction stages transition.we investigated the expression patterns of eyes absent 3 (EYA 3) and thyrotropin beta subunit (TSH β) genes in different tissues of seasonal estrous sheep(Sunite sheep, SNT)at different photoperiods (long photoperiod (LP) and short photoperiod(SP)) and year-round estrous sheep (Small Tail Han sheep, STH) at different reproduction stages (luteal phase and follicular phase) through q-PCR technology. The results showed that EYA 3 was widely expressed in different tissues of 2 breeds, while TSH β was expressed highly in pituitary. In most tissues, the expression levels of those 2 genes under long photoperiod (LP) were higher than those under short photoperiod (SP) in SNT, and the expression levels were higher at luteal phase than those at follicular phase in STH. When SP turned into LP in SNT, the expression levels of those 2 genes rose in pituitary, and EYA 3 showed a transient peak at LP 3, and the peak of TSH β was at LP 21. The expression of EYA 3 declined prior to TSH β. The results elucidated the expression characteristics of EYA 3 and TSH β in year-round estrous and seasonal estrous sheep, the expression characteristics of those 2 genes at different photoperiods and different reproduction stages in pineal and pituitary of sheep, which suggested that EYA 3 played an important role in pineal and might regulate seasonal estrous of sheep. In addition, EYA 3 and TSH β might be involved in the transition of estrous stages. When SP turned to LP, EYA 3 played a regulatory role earlier than TSH β.
Key words: sheep     EYA 3     TSH β     seasonal estrus     reproduction stage     expression pattern    

大部分绵羊品种具有季节性发情的特征,在长日照季节表现为乏情,短日照季节表现为发情。绵羊季节性发情导致羔羊肉不能实现四季均衡供应,严重制约了肉羊业的生产效率,因此,研究季节性发情调控机制以提高绵羊繁殖效率十分重要。目前关于该机制研究较为清楚的是动物通过甲状腺激素调节性腺生理活动进而实现对季节性发情的调控[1-2],这种机制由垂体结节部(Pars tuberalis, PT)促甲状腺细胞表达的两个长光照因子EYA 3与TSH β启动[3-7]

EYA基因编码一类眼缺失家族蛋白,目前已知该家族成员包含EYA 1、EYA 2、EYA 3和EYA 4,EYA因子具有磷酸酶活性,对于视网膜的发育具有重要调控作用[8-10]。其中EYA 3作为昼夜节律基因对绵羊季节性发情具有间接调控作用,其参与绵羊季节性发情长光照分子通路:绵羊将接收到的外界不同长度光照信号经下丘脑视交叉上核(Hypothalamic suprachiasmatic nuclei, SCN)传递至松果体,松果体可分泌承载光周期信号的褪黑素(Melatonin, MEL),MEL可与PT区域褪黑素受体(Melatonin receptor 1, MT 1)结合,长日照下,垂体分泌的EYA 3在天黑后12 h达到表达高峰,EYA 3表达峰在早上出现,MEL水平低,EYA 3峰值较高[11-13]。此机制启动了动物机体内部一系列识别长日照信号因子的分泌机制,进而间接调控绵羊性腺轴的关闭。EYA 3在不同物种组织中的表达情况已有相关研究报道,EYA 3可在绵羊垂体中表达,且在LP下表达量高于SP[14]EYA 3在大鼠下丘脑中表达,且LP下表达量也高于SP[15]EYA 3在人类的骨髓、胚胎、肝、脊髓、肌肉、大脑和肠中均有表达[10]。已知EYA 3可通过垂体间接调控性腺活动,但在不同光照条件和不同繁殖时期,EYA 3是否在绵羊垂体外的组织存在表达差异,并发挥调控作用,及由SP变为LP后,绵羊EYA 3表达变化趋势目前未见相关报道。

促甲状腺激素(Thyroid stimulating hormone, TSH)由糖蛋白α亚基(Glycoprotein hormone, alpha polypeptide, CGA)和β亚基(TSH-beta, TSH β)构成[16-17]TSH β可对光周期进行应答[3, 18-19],长光照时EYA 3表达升高,使PT分泌TSH β增多,TSH β与下丘脑基部的促甲状腺激素受体(Thyroid-stimulating hormone receptor, TSHR)结合介导了DIO 2的升高,DIO 2促使无活性的T4转化为有活性的T3,T3能够影响GnRH神经元的形态和空间位置,导致GnRH浓度释放产生差异,最终影响垂体激素的分泌,继而调控性腺的生理活动[20-23]。研究发现,TSH β在多种动物(大鼠、小鼠、猪、山羊、绵羊)的垂体中高表达[24-27];与EYA 3类似,在不同光照条件和不同繁殖时期,各组织中TSH β是否存在表达差异及由SP变为LP后绵羊TSH β表达变化趋势均鲜有报道。因此,本研究明确了上述两个基因在常年发情和季节性发情绵羊品种中的表达模式,并对各组织中两基因在不同光照条件下和不同繁殖时期间的表达差异进行研究,分析了由SP转变为LP后42 d内,EYA 3与TSH β在绵羊垂体中的表达变化趋势,研究结果有助于进一步揭示两个基因在绵羊发情性状中的作用。

1 材料与方法 1.1 试验材料 1.1.1 试验动物组织样品采集

不同时期目标基因组织表达谱试验:试验羊为天津市畜牧兽医研究所畜禽繁育基地人工控光条件下饲养的健康空怀苏尼特母羊;山东郓城小尾寒羊保种场的健康空怀小尾寒羊母羊。选取短光照(人工模拟配种季节)第21天和长光照(模拟休情季节)第49天的苏尼特羊成年母羊各3只,卵泡期和黄体期的小尾寒羊成年母羊各3只。屠宰绵羊后,迅速采集垂体、松果体、大脑、小脑、下丘脑、输卵管、子宫、卵巢、肾上腺和肾共10种新鲜组织样品,装入2 mL冻存管,置于液氮,之后储存在-80 ℃冰箱中备用。

季节性发情绵羊不同光照条件下,垂体中目标基因表达模式试验:苏尼特母羊在短光照(白天8 h:黑夜16 h)下,饲养42 d后,转至长光照(白天16 h:黑夜8 h)饲养49 d,并根据文献报道[11-12, 22, 28]与本实验室预试验结果,分别在SP 21、LP 3、LP 21、LP 33、LP 42共5个时间点(短光照第21天和长光照第3、21、33和42天)屠宰绵羊,每个时间点屠宰3只羊,迅速采集垂体组织置于液氮中短暂保存后,置于-80 ℃冰箱中保存备用。

1.1.2 试剂与仪器

RNAprep pure动物组织总RNA提取试剂盒(天根生化科技北京有限公司)、PrimeScriptTM RT Reagent Kit及SYBR@ Premix Ex TaqTM Ⅱ(TaKaRa, Japan)。PCR仪(T100型,美国Bio-rad)、超低温冰箱(美国Thermo)、电泳成像仪(Chemi Doc XRS+, 美国Bio-rad)、电泳槽(JY-SPFT,北京JUNYI)、离心机(5417型,德国Eppendorf)、荧光定量仪(罗氏480)。

1.2 方法 1.2.1 引物设计

根据NCBI数据库中绵羊EYA 3基因序列(GenBank登录号:NM_001161733)与TSH β基因序列(GenBank登录号:XM_004002368),用Primer 3软件各设计1对荧光定量引物,内参基因选用β-actin,引物信息见表 1

表 1 荧光定量PCR扩增引物序列 Table 1 Primer sequences of real-time PCR
1.2.2 反转录

使用PrimeScriptTM RT Reagent Kit反转录试剂盒合成cDNA第一链,按照说明书进行操作。反转录产物稀释后,用内参基因β-actin进行PCR检测,检测合格后,-20 ℃保存。

1.2.3 荧光定量PCR

用荧光定量PCR方法检测苏尼特羊与小尾寒羊不同组织及不同光照时间点苏尼特羊垂体中EYA 3与TSH β表达量,每个样品3个重复,以β-actin为内参基因,设置阴性对照。反应体系总体积为20 μL:SYBR Premix Ex Taq Ⅱ 10 μL,RNase Free ddH2O 6.4 μL,上、下游引物(10 pmol·L-1)各0.8 μL,cDNA 2 μL。反应程序:95 ℃预变性5 s;95 ℃变性5 s,60 ℃ 30 s,40个循环;反应结束后,进行熔解曲线分析。以2-ΔΔCT[29]计算目的基因相对表达量。

1.2.4 数据分析

使用SPSS13.0软件对数据进行统计分析。组间比较采用单因素方差分析(ANOVA)检验。P<0.05表示差异显著,P<0.01表示差异极显著。

2 结果 2.1 EYA3在SP和LP条件下SNT中的表达谱

荧光定量分析发现,EYA 3在苏尼特羊各组织广泛表达,且不同光照条件下,EYA 3均在松果体、输卵管和小脑中高表达,其中松果体表达量最高,其次是输卵管,然后是小脑,其它组织表达量较低,暗示EYA 3可能在绵羊松果体、输卵管和小脑组织中发挥作用;另外,LP下,EYA 3在各组织表达量均高于SP,且在松果体、大脑和输卵管中的表达量显著高于其它组织(P<0.05,图 1),暗示该基因在松果体、大脑和输卵管中的表达差异可能与季节性发情相关。

1~10.垂体、松果体、大脑、小脑、下丘脑、输卵管、子宫、卵巢、肾上腺和肾。下同。*.P<0.05 1-10.Pituitary, pineal gland, brain, cerebellum, hypothalamus, fallopian tube, uterus, ovary, adrenal gland and kindey.The same as below. *.P < 0.05 图 1 EYA 3在LP和SP下苏尼特羊不同组织中的表达水平 Figure 1 The expression levels of EYA 3 in different tissues of Sunite sheep under LP and SP conditions
2.2 EYA 3在黄体期和卵泡期STH中的表达谱

荧光定量分析发现,EYA 3也在小尾寒羊各组织广泛表达,且不同繁殖时期EYA 3均在垂体、松果体和小脑中高表达,且垂体和松果体中,黄体期高于卵泡期,但并无显著差异,其它组织表达量较低(图 2)。这一结果表明:除了垂体,小尾寒羊中EYA 3可能也在松果体和小脑中发挥作用。

图 2 EYA 3在小尾寒羊黄体期和卵泡期不同组织中的表达水平 Figure 2 The expression levels of EYA 3 in different tissues of Small Tail Han sheep at luteal phase and follicular phase
2.3 TSH β在LP和SP下,SNT中不同组织的表达

荧光定量分析发现,TSH β在苏尼特羊垂体中高表达,其它组织几乎不表达,且LP下垂体中表达量极显著高于SP表达量(P<0.01)(图 3)。

**.P<0.01。下同 **.P < 0.01.The same as below 图 3 TSH β在LP和SP下苏尼特羊不同组织的表达水平 Figure 3 The expression levels of TSH β in different tissues of Sunite sheep under LP and SP conditions
2.4 TSH β在STH黄体期和卵泡期不同组织的表达

荧光定量分析发现,TSH β在小尾寒羊垂体中高表达,其它组织几乎不表达,且黄体期垂体中表达量极显著高于卵泡期(P<0.01)(图 4)。

图 4 TSH β在小尾寒羊黄体期和卵泡期不同组织中的表达水平 Figure 4 The expression level of TSH β in different tissues of Small Tail Han sheep at luteal phase and follicular phase
2.5 SP转变为LP后,SNT垂体中,EYA 3与TSH β的表达变化模式

荧光定量分析发现,不同时间点苏尼特羊垂体中EYA 3与TSH β均在SP转至LP后表达量升高,其中EYA 3在LP 3达到表达高峰,之后逐渐降低;TSH β在LP 21达到表达高峰,之后逐渐降低。EYA 3表达量提前于TSH β开始降低,表明EYA 3发挥调控作用的时期早于TSH β,主要在LP 3之前;而TSH β主要在LP 21之前发挥调控作用。

SP 21、LP 3、LP 21、LP 33、LP 42分别代表短光照第21天和长光照第3、21、33和42天 SP 21, LP 3, LP 21, LP 33, LP 42 represent short photoperiod day 21, long photoperiod day 3, 21, 33 and 42 图 5 SP转变为LP后不同时间点苏尼特羊垂体中EYA 3与TSH β的表达模式 Figure 5 The expression pattern of EYA 3 and TSH β during the transition from SP to LP in pituitary of Sunite sheep
3 讨论 3.1 常年发情与季节性发情绵羊不同时期EYA 3和TSH β的表达谱分析

S.H.WOOD等[30]以绵羊为研究对象发现,EYA 3与TSH β均可在PT区域的促甲状腺细胞中表达,两基因通过TSH β-DIO 2-TH通路激活或抑制下丘脑-垂体-性腺轴,达到调控绵羊季节性发情[31-32]。本研究荧光定量结果表明,EYA 3与TSH β在小尾寒羊和苏尼特羊各组织中的表达情况基本一致,即在两品种绵羊中EYA 3均在多个组织中广泛表达,TSH β主要在垂体中高表达,表明TSH β主要在绵羊垂体中发挥作用。不同光照条件下,EYA 3基因在苏尼特羊松果体、输卵管、小脑中高表达,在小尾寒羊中,不同繁殖时期EYA 3在垂体、松果体和小脑中高表达,且黄体期表达量高于卵泡期;其中,黄体期EYA 3在松果体、大脑和输卵管中表达量显著高于卵泡期。结合已知的季节性发情调控网络涉及的上游部位(松果体、垂体和下丘脑),上述结果暗示,EYA 3可能也在松果体部位发挥作用并对绵羊季节性发情进行调控;另外,在绝大多数组织中EYA 3和垂体中TSH β表现为黄体期表达量高于卵泡期,暗示它们可能参与绵羊繁殖时期转换。

在苏尼特羊中,LP下TSH β在垂体中表达量极显著高于SP;小尾寒羊中,黄体期TSH β在垂体中表达量极显著高于卵泡期。季节性发情绵羊休情期PT区TSH β高表达,导致下丘脑基底部GnRH神经元末梢被室管膜细胞包围,与PT细胞接触减少,末梢释放GnRH的量也相应降低[30, 33]。据此,推测在小尾寒羊黄体期时,EYA 3表达量升高可促进垂体中TSH β高表达,进而通过TSH β-DIO 2-TH实现小尾寒羊下丘脑基底部GnRH神经元的作用,最终导致GnRH释放量降低;而卵泡期下丘脑基底部GnRH神经元末梢不被室管膜细胞包围,与PT细胞接触增多,导致GnRH释放量增加,促进FSH和LH的释放,从而影响卵泡发育和排卵,这一推测需进一步研究证实。

3.2 SP转至LP条件下,EYA 3与TSH β表达变化趋势

本试验通过对SP和LP不同时间点绵羊垂体中EYA 3及TSH β表达水平的检测,发现两基因表达趋势基本一致,均在SP转至LP后表达量升高。文献报道EYA 3与TSH β在绵羊昼夜节律调控中发挥重要作用[34-35]。K. H. Masumoto等[12]以雄性CBA/N小鼠为对象,通过PT区的全基因组表达谱分析发现了TSH β等长光照诱导基因,并对TSH β上游基因进行鉴定,发现EYA 3对TSH β有重要调控作用;在LP下,EYA 3可与Sine Oculis-related Homeobox 1 (Six 1)、促甲状腺胚胎因子(Thyrotroph embryonic factor, Tef)及肝白血病因子(Hepatic leukemia factor, Hlf)共同组成复合体与TSH β启动子结合促进TSH β的表达[11, 14, 36]。N. Nakao等[3]在对鹌鹑的光周期信号转换通路研究中发现,LP 1关灯14 h后,TSH β迅速表达,在接下来4 h后,DIO 2被快速诱导表达,并通过TSH β-DIO 2-TH通路最终影响垂体性腺轴激素的分泌[5-6]。鸟类中,在LP 1关灯后,上述两个基因高表达,但在LP 12以后表达量不再发生改变[15, 37]。本研究中,苏尼特羊垂体的检测结果表明,EYA 3在LP 3表达量最高,TSH β在LP 21表达量最高,提示EYA 3和TSH β分别主要在LP 3和LP 21之前发挥关键作用,启动机体内长日照机制,使绵羊成功进入长日照繁殖模式。

4 结论

本研究发现,LP下,EYA 3在苏尼特羊松果体中表达量显著高于SP,在小尾寒羊黄体期表达量均高于卵泡期,在两品种绵羊中TSH β均仅在垂体中高表达。结果表明,EYA 3可能同时在松果体部位发挥作用并对绵羊季节性发情进行调控,另外,这两个基因可能参与繁殖时期的转换。在SP转至LP过程中,EYA 3和TSH β分别主要在LP 3和LP 21之前发挥关键作用,启动长日照繁殖模式。

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