,四川农业大学,畜禽遗传资源发掘与创新利用四川省重点实验室,成都 611130Genetic regulation of broodiness in poultry
Lingqian Yin, Jinshan Ran, Jingjing Li, Peng Ren, Xianxian Zhang, Yiping Liu,Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China通讯作者:
编委: 李辉
收稿日期:2018-11-21修回日期:2019-03-20网络出版日期:2019-05-20
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Received:2018-11-21Revised:2019-03-20Online:2019-05-20
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作者简介 About authors
尹玲倩,硕士研究生,专业方向：家禽遗传育种与繁殖E-mail:
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尹玲倩, 冉金山, 李菁菁, 任鹏, 张贤娴, 刘益平. 禽类就巢性状的遗传调控[J]. 遗传, 2019, 41(5): 391-403 doi:10.16288/j.yczz.18-316
Lingqian Yin, Jinshan Ran, Jingjing Li, Peng Ren, Xianxian Zhang, Yiping Liu.
同大多数脊椎动物相比,禽类具有一种典型的繁殖特征,即就巢性。就巢行为是禽类为适应多变的环境,在漫长的自然选择中形成的一种利于繁衍后代和扩大群体规模的本能行为。这种行为主要表现在母禽进入产蛋窝巢的次数增加,同时伴随着停留时间延长,最终停止产卵而进行孵化。如果禽类长期处于典型就巢状态,会引起输卵管和卵巢发生萎缩,严重降低产蛋量甚至休产。姜润深等[1]根据鸡就巢时间和卧息时间长短,将鸡的就巢行为分为3种类型,即非就巢型、非典型就巢型和典型就巢型。影响禽类就巢的因素主要分为环境因素、内分泌因素和遗传因素。环境因素中光照和温度对就巢行为的影响较大,阴暗和高温环境会诱导禽类产生就巢行为。繁殖激素等内分泌因子直接参与禽类就巢性的调控,其中催乳素(prolactin, PRL)是引起和维持家禽就巢行为的主要激素。遗传因素是导致禽类产生就巢性的根本原因,对于就巢性的遗传规律有多种解释,但最终证实控制就巢性的基因位于常染色体上,其中有研究推测白来航鸡PRL基因启动 子中的一段24 bp的纯合插入序列可能会影响白来航鸡体内PRL的表达,进而影响白来航母鸡的就 巢性[2,3]。
与产蛋期母禽相比,就巢期母禽的繁殖行为和生殖系统的形态都发生了显著变化。就巢期间禽类的卵巢出现了严重的萎缩与退化,无等级卵泡发育或极少数发育的等级卵泡停止发育并出现闭锁,卵泡中颗粒细胞层和膜细胞显著减少[4,5,6,7]。深入研究发现,造成就巢期禽类卵巢形态发生改变的直接原因是由于禽类体内的内分泌激素的含量发生改变,其中主要是繁殖激素含量的改变。通过对就巢期禽类血清中的繁殖激素进行测定,发现处于就巢期的鸡、鸭、鹅和火鸡(Meleagris gallopavo)的血清中PRL含量显著高于产蛋期[5,8,9]。此外,雌二醇(estradiol, E2)、孕激素(progesterone, P4)、促卵泡生成素(follicle-stimulating hormone, FSH)和促黄体生成素(luteinizing hormone, LH)等繁殖激素同样也被证实参与禽类就巢性的调控[10,11]。除繁殖激素外,许多神经内分泌因子,如催乳素释放因子血管活性肠肽(vasoactive intestinal polypeptide, VIP)[12]、多巴胺(dopamine, DA)和5-羟色胺(5-hydroxytryptamine, 5-HA)[13,14]等,也能够通过影响PRL的合成与释放间接调控禽类的就巢行为。
尽管通过主动、被动免疫和环境调控可以在一定程度上降低就巢行为的发生率,但这种方法并不适用于现代化大规模的禽业生产。因此,从根本上消除禽类的就巢性是目前亟待解决的问题。开展就巢性相关候选基因的研究有助于确定控制就巢性状的主效基因,深入了解就巢性的遗传基础和具体的分子机制,最终通过遗传育种的方法培育无就巢性的禽类品种,以此来提高家禽的繁殖性能。近几年在环境和内分泌研究的基础上,研究人员从分子层面进一步揭示了调控禽类就巢性的因素,并对抑制或消除就巢性的方法进行探索。本文根据前人对禽类就巢性的研究,对参与调控禽类就巢性的基因、miRNA和信号通路进行了综述。
1 参与就巢性调控的基因
1.1 繁殖激素相关基因
生殖激素含量的变化是诱导禽类产生就巢性的直接原因。最初,研究主要集中于PRL、FSH和LH等繁殖激素相关基因的表达和多态性的分析。Jiang等[2]在探究绿壳蛋鸡中PRL和PRLR基因的变异对繁殖性状的影响时发现,PRL基因启动子区域的PRLpro2位点的插入/缺失突变对平养绿壳蛋鸡的就巢性有显著的影响。朱盼[15]利用荧光定量PCR技术检测了在4个时期中皖西白鹅垂体、下丘脑、卵巢3个组织中PRLR基因的表达水平,发现PRLR基因在就巢期的表达量最高,且在就巢前期的表达量达到最大,卵巢中PRLR基因的表达水平显著高于下丘脑和垂体。但是,段修军等[16]研究发现PRLR在就巢期的垂体中表达量高于卵巢和下丘脑。这可能是由于禽类所处的就巢期阶段不同和物种差异所导致。此外,PRL基因的不同单倍型所表现出来的就巢行为也存在差异。梁勇等[17]发现催乳素基因内含子2的第35碱基(T/C)位点、内含子4的第1824碱基(A/G)位点的突变会导致家鸡群体表现出不同的就巢率。除PRL和PRLR外,其他繁殖激素基因也被证实参与禽类就巢性的调控。吴国平[18]对浙东白鹅繁殖周期中PRL、FSH和LH基因的表达进行了分析,发现FSHβ和LH的表达量在就巢期的浙东白鹅垂体中显著下调表达。张响英等[19]检测了处于不同繁殖周期的狮头鹅3种组织中促卵泡素β亚基(follicle- stimulating hormone β, FSHβ)和促卵泡素受体(follicle-stimulating hormone receptor, FSHR)基因的表达水平,结果表明FSHβ和FSHR在产蛋期的相对表达量极显著高于就巢期和休产期,由此推测FSHβ及FSHR表达水平的变化与鹅的繁殖周期密切相关。Wu等[20]对GnRH的研究也得到了相似的结果,就巢期番鸭下丘脑、垂体和卵巢等组织中的GnRH表达量显著低于产蛋期。在禽类不同的繁殖周期中,GnRH、LH、FSHβ和FSHR基因具有相似的表达模式,而PRL和PRLR具有相反的表达模式。因此推测就巢禽类的部分组织中GnRH、LH、FSHβ和FSHR的表达会受到PRL和PRLR基因的抑制性调控,从而诱导就巢行为的发生;反之,在非就巢期禽类的体内GnRH、LH、FSHβ和FSHR的表达量上调则会抑制PRL和PRLR基因的表达,从而抑制禽类的就巢行为的发生。综上所述,可以发现调控禽类繁殖行为的GnRH、LH、FSHβ、FSHR、PRL和PRLR基因都是参与就巢性调控的候选基因。
1.2 其他相关基因
就巢性是一种受多基因控制的复杂性状,除了繁殖激素相关基因外,研究者还发现了其他参与就巢性调控的基因,如多巴胺D1/D2受体基因(dopamine D1/2 receptor, DRD1/2)、血管活性肠肽(vasoactive intestinal polypeptide, VIP)及其受体(vasoactive intestinal polypeptide receptor, VIPR)基因等。DRD1和DRD2被证实参与了禽类繁殖行为的调控。Xu等[21]在探究DRD1基因及其单倍型对鸡蛋产量和就巢性状的影响时,发现DRD1基因4个SNP位点变异与就巢频率显著相关,且DRD1基因在非就巢鸡的皮下和腹部脂肪中的表达显著高于处于就巢期的鸡。DRD2基因中的两个位点A-16105G和T+619C也被认为与鹅的就巢行为显著相关,A-16105G 主要与就巢频率相关,而T+619C则参与调控就巢行为持续时间的长短[22]。此外,有研究表明VIP及VIPR可能也是调控鸡就巢性的候选基因。对斑胸草雀(Taeniopygia guttata)就巢性的研究中发现,当雌性和雄性斑胸草雀处于就巢的情况下,其大脑中的VIP转录活性增强[23]。Zhou等[24]发现VIPR-1基因序列中的C+598T和C+53327T的变异与禽类的就巢性显著相关,前者主要与就巢频率有关,而后者主要控制就巢持续时间的长短。相关的研究也表明VIP基因的表达与多巴胺受体基因之间存在相互作用关系,VIP和PRL的分泌受下丘脑刺激性DRD1和抑制性DRD2的双重作用[25,26]。除上述4种基因外,Shen等[27]通过抑制性消减杂交技术(suppressive subtractive hybridization, SSH)分析证实GTP酶激活Rap/RanGAP域样-1(GTPase activating Rap/RanGAP domain-like 1, GARNL1)也是参与鸡就巢性调控的候选基因,GARNL1在就巢期的鸡下丘脑和垂体中的表达水平显着高于非就巢的鸡。Xu等[28]通过转录组测序发现胆固醇侧链裂解酶(cholesterol side-chain cleavage enzyme, CYP11a)和多巴胺β-羟基酶(dopamine beta-hydroxylas, DBH) 基因在就巢期的禽类卵泡中高度表达,这两个基因可作为参与就巢性调控的候选基因。Liu等[29]通过比较就巢初期和产蛋期的鹅的下丘脑、垂体、卵巢基质和非等级卵泡壁的转录组,发现催产素-神经生长素(oxytocin-neurophysin, OXT)、脊索生成素样蛋白1 (chordin-like protein 1, CHRDL1)和生长激素(growth hormone, GH)可能是参与就巢性调控的新候选基因;此外,还发现下丘脑分泌素(hypocretin, HCRT)和阿黑皮素原(pro-opiomelanocortin, POMC)这两个与食欲调控相关的基因在就巢初期鹅的下丘脑组织中显著差异表达,因此推测就巢初期食欲减退可能是禽类就巢行为的第一步表现,同时也是引发鹅产生就巢行为的决定性因素。Liu等[7]利用全转录组技术比较分析就巢期和产蛋期鸡的卵巢组织,筛选出大量与就巢性相关的基因：如与繁殖过程相关的基质金属肽酶2 (matrix metallopeptidase 2, MMP2)、细胞色素P450家族1亚家族B成员1 (cytochrome P450 family 1 subfamily B member 1, CYP1B1)、多巴胺β-羟化酶(dopamine beta-hydroxylase, DBH)、抑制素β A/B(inhibin beta A/B, INHβA/B)和催产素受体 (oxytocin receptor, OXTR)基因等;与细胞增殖凋亡相关的Smad家族成员2 (Smad family member 2, SMAD2)、p38丝裂原活化蛋白激酶(p38 mitogen- activated protein kinases 11, MAPK11)、S期激酶相关蛋白2 (S-phase kinase-associated protein 2, SKP2)、受体相互作用蛋白激酶-1(receptor-interacting protein kinase-1, RIPK1)、生长停滞和DNA损伤诱导45 (growth arrest and DNA damage-inducible 45, GADD45)基因和Caspase基因家族。
此外,褪黑素受体(melatonin receptor 1C, Mel-1c)[30]、抗缪勒氏管激素受体Ⅱ (Anti-Müllerian hormone receptor Ⅱ, AMHRII)[31]、雷帕霉素靶蛋白基因(mechanistic target of rapamycin, mTOR)[32]、核受体辅激活蛋白1 (nuclear receptor co-activator 1, NCOA1)、胆固醇调节元件结合蛋白2 (sterol regulatory element binding transcription factor 2, SREBF2)、Ral GTP酶活化蛋白亚基α-1 (Ral-GTPase activating protein α-1, RALGAPα1)[33]和垂体特异转录因子(POU- domain family of transcriptional activators, Pit-1)[34]等基因也被证实参与禽类就巢性的调控(表1)。
Table 1
表1
表1 家禽就巢性相关基因及其信号通路
Table 1
| 候选基因 | 候选microRNA | 相关信号通路 |
|---|---|---|
| PRL[2,5,8,9]、MAPK11[7]、SKP2[7]、RIPL1[7]、GADD45[7]、INHβA/B[7]、CYP1B1[7]、MMP2[7]、OXTR[7]、DBH[7]、LH[10,11]、VIP[12]、PRLR[15,16]、 FSHβ[18,19]、GnRH[20]、SMAD[7,42,61,70~73]、DRD1[21,22]、VIPR[23,24]、DRD2[25,26]、GARNL1[27]、CYP11a[28]、HMOX1[29]、FOS[29]、HSP90AA[29]、CDK1[29]、HCRT[29]、POM6C[29]、CHRDL1[29]、GH[29]、OXT[29]、HSP90[30]、Mel-1c[30]、AMHRII[31]、mTOR[32]、NCOA1[33]、SREBF2[33]、RALGAP α-1[33]、Pit-1[34]、GHR[36]、 HSD3B2[62]、GAB[68]和GDF9[71] | miR-34家族[7]、miR-18[7]、miR-98[7]、miR-128[7]、miR-135[7]、miR-148[7]、miR-320[35,40,46,47]、miR-202[35,44]、miR-146家族[36]、miR-143家族[36]、miRn1[36]、miRn10[36]、miRn11[36]、miRn12[36]、miR-199[37]、miR-30家族[37]、miR-132[38,39]、miR-378[41]、miR-224[42,48]、miR-21[52]、miR-204[53]、miR-205[54,55] 和miR-10家族[37,57,58] | p53信号通路[7]、卵母细胞减数分裂[28]、钙信号通路[28]、GnRH信号通路[35]、类固醇激素生物合成[35]、Wnt信号通路[37]、TGF-β信号通 路[35,70,71]、激素反应[78]、卵泡发育[78]、自噬氧化通路[78]和黄体酮信号通路[78] |
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2 参与就巢性调控的microRNAs
近期研究表明,microRNAs(miRNAs)参与哺乳动物的卵巢性腺发育、类固醇生成、细胞凋亡、排卵和禽类就巢性等过程的调控(表1)。Xu等[35]通过Solexa测序技术筛选出大量与禽类繁殖相关的miRNAs,其中G-miR-320、G-miR-202、G-miR-146和G-miR-143*被证实参与禽类就巢行为的调控。Chen等[36]利用高通量测序技术比较不同繁殖时期鹅的卵巢组织,也证实G-miR-146和G-miR-143*参与禽类就巢性的调控;此外,还发现let-7家族在两个测试组中表达量最丰富,其次是miR-146家族和miR-143家族;在两组卵巢组织中还筛选出4个新的差异表达的miRNAs,分别是miRn1、miRn10、miRn11和miRn12,且miRn1仅在就巢组中表达;对新的miRNAs进行功能预测分析,发现miRn10和miRn11参与卵巢中PRLR的转录调控。Yu等[37]也发现let-7、miR-10、miR-30和miR-199家族参与不同时期鹅卵泡的发育,并推测这4个miRNA家族是调控卵泡发育过程中的核心因素。Liu等[7]利用全转录组测序筛选出大量与就巢性相关的非编码RNA,如p53信号通路中的重要调控因子miR-34b 和 miR-34c,参与卵巢类固醇合成调控的miR-18、miR-98、miR-128、miR-135和miR-148等。综上所述,参与繁殖活动调控的miRNAs主要分为两类：一类参与就巢期卵巢内繁殖激素调节途径;另一类调控卵巢内体细胞和生殖细胞的增殖与凋亡。2.1 参与卵巢类固醇激素合成的microRNAs
卵巢中类固醇激素的水平与卵巢上卵泡的发育状态和排卵有着直接的联系,类固醇激素的合成受到许多酶和转录因子的调控。近年来的研究也证实miRNAs在调控卵巢类固醇合成中扮演着重要的角色。作为一个多功能的miRNA,miR-132被证实参与调控禽类卵巢中类固醇激素的合成。Sang等[38]发现miR-132在多囊卵泡综合征患者的卵泡液中下调表达,并且参与卵泡中雌二醇的合成。Wu等[39]通过向体外培养小鼠的原代颗粒细胞转入miR-132模拟物,发现其能够显著上调颗粒细胞中雌激素的浓度,进一步研究表明,miR-132主要是通过结合其靶基因孤儿核受体1 (neural orphan nuclear receptor 1, Nurr1)基因,抑制Nurr1的表达,进而减少Nurr1对细胞色素P450 19A1 (cytochrome P450 19A1, Cyp19a1)基因的抑制作用,促进雌二醇的生物合成。miR-320也被证实参与调控卵巢类固醇激素的合成。Zhang等[40]研究miR-320的表达对多囊卵巢综合征患者卵丘细胞中雌二醇含量的影响,发现miR- 320主要是通过miR-320a/RUNX2/CYP11A1 (CYP19A1)级联系统来调控卵丘细胞中胰岛素样生长因子1 (insulin-like growth factor 1, IGF1)基因诱导的孕酮和雌二醇产生,抑制miR-320a表达会显著降低正常卵丘细胞中孕酮和雌二醇产生。Sang等[38]为了探究人卵泡液中与类固醇合成有关的miRNAs,将选择的12个miRNAs类似物和抑制剂转染到体外培养的人卵巢颗粒样肿瘤细胞(steroidogenic human granulosa- like tumor cell line, KGN)中,发现miR-320类似物能够促进雌二醇的分泌,添加其抑制剂会导致雌二醇分泌降低。
此外,也有研究表明miRNAs可以通过调控颗粒细胞中芳香化酶基因的表达来影响雌二醇的合成。Xu等[41]发现miR-378通过与芳香化酶基因的3°-UTR中的两个结合位点结合,干扰芳香化酶的正常功能,以此来减少猪卵泡颗粒细胞中雌二醇的产生。Yao等[42]的研究则发现miR-224在转化生长因子β 1 (transforming growth factor beta, TGF-β1)处理的小鼠腔前颗粒细胞(granulosa cell, GC)中显著上调表达,miR-224可以靶向结合Smad4来增强TGF-β1诱导的GC细胞增殖,并且miR-224和TGF-β1均可通过增加CYP19A1的转录水平促进GC细胞释放雌二醇。
2.2 参与卵巢细胞增殖和凋亡的microRNAs
处于就巢期的禽类,其生殖器官在功能上显著退化。先前的研究不仅证实了就巢期禽类的卵巢中类固醇生成减少,也发现在就巢期禽类的卵巢中细胞增殖和凋亡过程显著减缓[7]。大量研究表明,miRNAs参与许多通路中细胞增殖和凋亡基因的表达调控。miR-202主要在脊椎动物的性腺中表达,近期关于miR-202的研究主要集中在其对肿瘤细胞增殖的调控方面,证实miR-202是多种疾病早期诊断的标志物。此外,也有研究表明miR-202参与繁殖相关活动的调控。Xu等[35]的研究发现,miR-202在就巢期鹅的卵泡中显著上调表达。Zhang等[43]发现miR-202-5p主要在斑马鱼(Barchydanio rerio var)的卵母细胞中表达和积累。Gay等[44]利用CRISPR/ Cas9技术对青鳉(Oryzias latipes)体内的miR-202进行敲除,发现miR-202缺失会损害早期卵泡的发育,降低卵巢中成熟卵泡的数量和质量,证实miR-202是参与调节雌性卵泡募集和生长的关键miRNA。除了参与卵泡的生长发育调控,miR-202还能协同雌激素调控鸡胚胎的性别分化,miR-202的上调与鸡胚性腺中的雄性睾丸的分化一致[45]。miR-320在调控就巢期禽类卵泡颗粒细胞的增殖和凋亡中也发挥了重要的作用。有研究发现miR-320在就巢期的鹅卵巢中的表达量显著低于产蛋期[35]。Feng等[46]研究发现,在分裂细胞数量较多的早期胚胎中,miR-320的表达量显著高于分裂细胞数量较少的早期胚胎,敲除小鼠卵母细胞中mmu-miR-320会显著抑制卵母细胞的进一步分裂。也有研究发现miR-320在调控颗粒细胞的功能中发挥着重要的作用。如Yin等[47]发现miR-320主要在发育阶段的小鼠卵泡颗粒细胞和卵母细胞中表达;此外,miR-320可以通过靶向结合转录因子E2F1和SF-1蛋白调节卵泡发育中颗粒细胞的增殖。
除了上述两种miRNAs,有研究证实miR-224[48]、miR-26a-5p[49]、miR-148b[50]和miR-214[51]等也参与卵巢中卵泡的发育和颗粒细胞、膜细胞的增殖调控。近期的研究发现,大量与细胞凋亡和自噬相关的miRNAs在就巢期和产蛋期的禽类下丘脑、卵巢和卵泡中差异表达,如miR-21[52]、miR-204[53]、miR-205[54]、G-miR-34b-5p、miR-6006和G-miR-1620[7]等。
miR-205是一种高度保守的miRNA,能够通过多条途径调控细胞的凋亡。Tian等[55]发现miR-205可以直接靶向结合抗凋亡基因Bcl-2,通过调控Bcl-2的表达来抑制细胞的增殖,诱导细胞凋亡。Zhang等[56]证实miR-205在患慢性肾病小鼠的肾细胞中显著下调表达,通过体内、体外实验证实miR-205通过与趋化因子样因子(CKLF)样MARVEL跨膜结构域家族成员4 (Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family, CMTM4) mRNA的3'UTR结合,调控CMTM4基因的表达,进而抑制细胞的凋亡。此外,也有研究证实miR-205参与繁殖活动的调控。如Zhang等[54]研究miR-205在小鼠颗粒细胞(mGCs)中的表达,发现在闭锁的卵泡中miR-205的表达量显著增加,环腺苷一磷酸反应元件结合蛋白1 (cyclic adenosine monophosphate responsive element binding protein 1, CREB1)是其直接的靶向基因;同时发现过表达miR- 205可以通过改变小鼠卵泡中促凋亡因子caspase-3/9的活性来促进细胞凋亡。
另一个高度保守的miR-10家族也被证实参与细胞的增殖和凋亡。Yu等[37]研究发现,miR-10家族成员在就巢期和产蛋期的鹅卵泡中高度表达。方芳等[57]发现miR-10b能够靶向结合脑源性神经营养因子(brain-derived neurotrophic factor,BDNF),通过抑制BDNF的表达来抑制山羊卵巢颗粒细胞的活性。Tu等[58]在探究miR-10家族成员对卵泡发育的调控作用时,发现miR-10a和miR-10b在卵泡成熟过程中表达量逐渐减少,但在卵泡闭锁时期表达量增加;通过进一步研究发现,miR-10a和miR-10b能通过抑制BDNF和TGF-β途径中的关键因子的表达来抑制卵泡中颗粒细胞的增殖,诱导颗粒细胞凋亡;此外,FSH和FSH介导因子—环腺苷一磷酸(cyclic adenosine monophosphate, cAMP)能够抑制颗粒细胞中miR-10a和miR-10b的表达,从而降低miR-10a和miR-10b对颗粒细胞增殖的抑制。研究证实FSH在就巢期禽类的卵巢中含量显著低于产蛋期[5,10,11],因此推测就巢期FSH含量的降低减少了对miR-10a和miR-10b的抑制,导致卵泡颗粒细胞增殖受到抑制。
综上所述,miR-202、miR-320家族、miR-205家族和miR-10家族在调控就巢期禽类卵巢生理功能中发挥了重要作用。
3 参与就巢性调控的相关信号通路
禽类的就巢性是一个受多种因素调控的复杂性状,其中繁殖激素含量的改变和卵巢功能退化是就巢期禽类的典型特征。从产蛋期进入就巢期,该过程涉及多条信号通路的参与(表1)。大量的研究证实,参与禽类就巢性调控的基因主要富集于细胞周期、细胞增殖和凋亡通路。Liu等[7]利用全转录组技术比较分析就巢期和产蛋期鸡的卵巢组织,通过GO和KEGG富集分析,发现差异表达的基因主要富集于PI3K-Akt信号传导、cGMP-PKG信号传导、FoxO信号传导、细胞周期、Hippo信号传导、MAPK信号传导、细胞凋亡和TGF-β信号传导等途径;其中大部分通路参与调控细胞的增殖和凋亡。除上述的细胞增殖和凋亡通路外,部分差异表达的基因还富集在与繁殖相关的通路上。Xu等[35]通过Solexa测序技术筛选出大量的与繁殖和就巢性相关的miRNAs,通过对靶基因的功能进行富集分析,发现基因富集最多的通路是繁殖通路,包括TGF-β信号通路、GnRH信号通路和类固醇激素生物合成信号通路。3.1 类固醇激素的生物合成
就巢期禽类的卵巢高度萎缩,颗粒细胞和膜细胞数量减少,导致卵泡内合成的类固醇激素的含量降低。雌二醇和孕酮是参与禽类就巢性调控的两种主要的类固醇激素,由卵泡的颗粒细胞和膜细胞合成,这一过程主要受FSH的调控。FSH可以通过FSH-cAMP-蛋白激酶A (protein kinase A, PKA)通路调控颗粒细胞中类固醇激素的合成。FSH与膜上的G蛋白偶联受体结合,激活下游的腺苷酸环化酶和PKA信号转导途径,调控参与雌二醇和孕酮合成的基因转录水平,影响卵泡中类固醇激素的合成。Cui等[59]在体外培养的3T3-L1细胞中添加FSH,发现能够提高细胞中雌二醇的含量,同时检测细胞中胆固醇、孕酮含量和cAMP-PKA通路以及过氧化物酶体增殖物激活受体(peroxisome proliferators-activated receptor, PPAR)途径中的相关基因的表达量,证实在体外培养的3T3-L1细胞中,FSH能够通过激活cAMP- PKA通路,活化下游CREB基因与过氧化物酶体增殖物激活受体γ (peroxisome proliferators-activated receptor γ, PPARγ)的结合,促进胆固醇向雌激素的转化。此外,FSH还可以通过FSH-P38 MAPK-StAR/ P450arom通路调控颗粒细胞中类固醇激素的生成。Yu等[60]研究发现,FSH通过两条不同的途径诱导颗粒细胞中雌激素和孕酮的生成,FSH激活P38MAPK通路,P38MAPK激活下游的孤儿受体家族的肝脏受体同源物-1 (liver receptor homolog-1, LRH-1)基因,调控芳香化酶家族P450arom的表达,进而影响颗粒细胞中雌激素的合成;此外,由FSH激活的P38MAPK途径能够诱导下游另一个孤儿受体基因(orphan receptor-1, DAX-1)的活化,进而影响类固醇生成急性调节蛋白(steroidogenic acute regulatory protein, STAR)基因的转录水平,调控颗粒细胞中孕酮的合成。FSH诱导的TGF-β信号通路也被证实参与颗粒细胞中类固醇激素生成的调控。Liang等[61]研究证实FSH能够协同TGF-β通过Smad途径调控颗粒细胞中雌激素的合成;此外,TGF-β3还可以协同SF-1来调控Cyp19a1 的表达,最终影响颗粒细胞中雌激素的释放。Lai等[62]发现FSH也能够通过激活颗粒细胞中的TGF-β1,诱导钙调神经磷酸酶介导的去磷酸化,激活cAMP反应元件结合蛋白CREB,调节转录共激活因子2 (CREB regulated transcription coactivator 2, CRTC2)的活性,而CRTC2则参与调节STAR、Cyp11a1和羟基δ5类固醇脱氢酶3β(hydroxy delta 5 steroid dehydrogenase 3 beta, HSD3B)的表达,促进颗粒细胞中孕酮的产生。
叉头转录调节因子2 (forkhead transcriptional regulator 2, FOXL2)是一种基因转录因子,在禽类卵泡中主要参与颗粒细胞功能的调控。有研究表明,FOXL2能够诱导颗粒细胞中雌激素的合成。Wang等[63]的研究发现,FOXL2通过两条途径激活芳香酶基因Cyp19a1的转录,调控罗非鱼(Oreochromis spp)卵巢中类固醇激素的合成,其中Foxl2可以直接通过其叉头结构域与Cyp19a1基因的启动子结合,激活Cyp19a1的转录;此外,Fox12也可以通过叉头结构域与Ad4BP/SF-1的配体结构域结合,形成异二聚体,并增强Ad4BP/SF-1介导的Cyp19a1转录。毛岩等[64]探究DNAJ同源亚家族B成员11 (DNAJ homolog subfamily B member 11, DNAJB11)与FOXL2在调控小鼠卵泡颗粒细胞中雌激素合成中的互作关系,发现FOXL2能够靶向结合DNAJB11基因,增强FOXL2蛋白的稳定性,促进下游FOXL2诱导的Cyp19a的转录活性,从而增强颗粒细胞中雌二醇的合成。Li等[65]也证实FOXL2在雌激素合成中发挥了重要的作用,敲除雌性罗非鱼FOXL2基因后,罗非鱼体内的卵母细胞表现出不同程度的变性,血清雌二醇-17β水平和芳香酶基因Cyp19a1a表达显著降低;因此,推测FOXL2可能通过调控芳香酶Cyp19a1a基因的表达来影响雌激素的合成。Folger等[66]发现可卡因和苯丙胺调节转录物(cocaine- and amphetamine-regulated transcript, CART)信号通路也参与了不同发育阶段卵泡颗粒细胞中雌激素的调控,抑制CART信号传导会影响发育后期的卵泡中雌二醇合成;此外,在颗粒细胞的体外培养中添加CART成熟肽,能够减少FSH刺激的体外培养颗粒细胞中雌二醇的产生。
3.2 细胞增殖与凋亡
众多转录组学的研究结果显示,在就巢期和产蛋期的禽类卵巢中差异表达的基因主要富集于细胞周期、细胞增殖和凋亡通路。这与就巢期禽类卵巢的形态学观测结果相符,与产蛋期相比,就巢期禽类的卵巢体积显著萎缩,卵巢表面无等级卵泡的发育。Yu等[37]对差异表达的miRNA靶基因进行通路富集分析,发现除了报道较多的繁殖通路显著富集外,在卵泡中还存在显著富集的细胞增殖和凋亡通路,如MAPK信号通路、细胞因子—细胞因子受体相互作用,以及卵泡发生中的Wnt信号通路等。Liu等[7]研究发现,就巢期萎缩卵巢中下调的基因主要富集在PI3K-Akt信号通路、ECM受体相互作用、Jak-STAT信号通路和TGF-β信号通路中,而在就巢期萎缩卵巢中上调的基因则主要富集于类胆碱突触、吞噬体、Notch信号通路和视黄醇代谢通路。PI3K-Akt信号通路在调控细胞增殖、迁移、凋亡和代谢等多种生理活动中发挥着重要的作用。研究表明,PI3K-Akt通路参与调控卵巢和卵泡中细胞的增殖。高雪等[67]探究结缔组织生长因子(connective tissue growth factor, CTGF)对颗粒细胞增殖的影响,发现抑制CTCF基因的表达会降低PI3K、P-AKT蛋白的表达,进而抑制颗粒细胞的增殖,并诱导其发生凋亡。因此,推测CTCF可能是通过PI3K/AKT信号通路来发挥其调控颗粒细胞增殖和凋亡的功能。先前的研究也证实PI3K/AKT通路是转导FSH信号的关键途径。如Hunzicker-Dunn等[68]利用FSH刺激体外培养的大鼠颗粒细胞,并探究PKA和生长因子受体结合蛋白相关蛋白2 (growth factor receptor-bound protein-associated binding protein 2, GAB2)基因在卵巢颗粒细胞中对FSH刺激的PI3K/AKT信号传导途径的作用,发现FSH刺激细胞后,激活PKA和下游的胰岛素受体底物-1 (insulin receptor substrate-1, IRS-1)、GAB2的表达;GAB2作为PKA的锚定蛋白,能够促进PKA与IRS-1复合物的形成,增强IRS1对下游PI3K/AKT信号途径的激活作用,最终起到促进细胞增殖,抑制细胞凋亡,增强类固醇和蛋白质激素基因转录、翻译和启动颗粒细胞分化的作用。Law等[69]的研究充分阐明了FSH刺激的G蛋白偶联受体(G protein-coupled receptors, GPCRs)-cAMP- PKA-PP1/MYPT1-IGF/IGFIR/IRS1-PI3K/AKT通路在调控颗粒细胞增殖中的反应机制,利用FSH刺激体外培养的腔前颗粒细胞,发现FSH可以通过GPCR直接激活PKA和蛋白磷酸激酶1 (protein phosphatase 1, PP1)的表达;此外,激活的PKA还可以通过促进肌球蛋白磷酸酶靶向亚基1 (myosin phosphatase targeting subunit 1, MYPT1)的磷酸化来进一步激活PP1;激活的PP1cβ/MYPT1能够促进下游IRS1丝氨酸多位点去磷酸化和IRS1 YXXM的磷酸化,进而激活PI3K/AKT信号途径,促进颗粒细胞的增殖。这种机制不仅存在于卵巢腔前颗粒细胞中,同样也存在于排卵前的颗粒细胞中。就巢期的禽类卵巢内,颗粒细胞数量锐减,FSH的含量也显著降低。因此,FSH诱导的PI3K/AKT信号途径受到抑制,最终导致颗粒细胞的增殖和类固醇的合成受到抑制。
TGF-β通路不仅参与卵巢颗粒细胞中类固醇激素的合成,同样也被证实参与调控卵巢内细胞的增殖和凋亡。TGF-β信号通路主要通过激活下游的接头蛋白SMADs和不同miRNAs的表达,参与卵巢颗粒细胞的增殖和凋亡调控。Du等[70]探究FSHR在滤泡闭锁中的作用机制,发现TGF-β/SMAD4-miR- 143-FSHR信号途径在调控颗粒细胞凋亡中发挥重要的作用,激活TGF-β信号途径,能够促进下游的SMAD4接头蛋白的表达,SMAD4能够靶向结合miR-143,下调miR-143的表达,进而降低miR-143对FSHR基因的转录抑制作用,减少miR-143诱导的GC细胞凋亡。除上述在经典TGF-β通路中发挥正向调控作用的SMAD4蛋白外,SMAD7蛋白也能够通过负反馈调控的方式参与TGF-β信号通路对细胞周期的调控。Gao等[71]证实在体外培养的小鼠颗粒细胞中,TGF-β1、骨形态发生蛋白4 (bone morphogenetic protein 4, BMP4)和生长转化因子9 (growth differentiation factor 9, GDF9)基因能够促进SMAD7的表达,而SMAD7则可以通过与TGF-β1靶向结合,降低TGF-βR1-SMAD2/3信号传导的活性,负反馈调控TGF-β信号通路对细胞增殖的作用。近期的研究发现,SMAD7能够通过调控TGF-βR1的转录活 性,调控TGF-β信号传导途径,增强猪颗粒细胞的凋亡;但miR-181b和miR-92a则可以靶向结合SMAD7,减弱SMAD7对TGF-β信号传导途径的调控作用,抑制GC凋亡[72,73]。
Notch信号通路在胞间信号传导和细胞形态发生中发挥重要的作用,主要是通过受体Notch1-4与DSL (Delta/Serrate/lag-2)蛋白结合,将信号传递给下游的螺旋-环-螺旋转录因子(helix-loop-helix transcription factors, Hes1), myc原癌基因家族(myc protooncogene family, MYC)等信号分子,发挥调控细胞生理功能的作用。近期有研究表明,Notch信号通路在早期卵泡的发育和胚胎的形成中发挥着重要的作用,影响细胞的增殖,分化和凋亡。Irles等[74]发现Notch信号通路能够维持德国小蠊(Blattlla germanica)的卵泡处于一种既不发育也不凋亡的稳定状态。Trombly等[75]探究Notch信号传导对原始卵泡形成的影响,发现Notch2及其配体基因Jagged1和下游靶基因Hes1、Hey2等在新生小鼠的卵巢中表达,在体外卵巢培养系统中抑制Notch信号通路,发现卵巢上原始卵泡的百分比显着降低,而生殖细胞的百分比显着增加。Jing等[76]利用DAPT抑制山羊卵泡细胞中的Notch信号途径,显著降低了卵泡中颗粒细胞的数量,同时阻遏Notch信号途径也会降低芳香酶基因的表达,进而影响雌二醇的合成。同时还发现Notch信号通路主要是通过调节与细胞凋亡相关基因的表达,进而调控颗粒细胞的发育。此外,在近期的研究中也发现Notch信号通路协同P13K/ Akt、MAPK和ERK通路共同调控颗粒细胞的增殖、凋亡和分化。Prasasya等[77]发现锯齿状典型Notch配体1 (jagged canonical Notch ligand 1, JAG1)是小鼠卵巢中表达最多的Notch配体,敲除JAG1和免疫球蛋白κJ区域的重组信号结合蛋白基因(recombination signal binding protein for immunoglobulin kappa J region, RBPJ),会抑制颗粒细胞的分化,降低颗粒细胞中的类固醇合成因子和相关酶的表达量,影响类固醇的合成和分泌;而抑制JAG1的表达能够促进颗粒细胞的增殖。
4 结语与展望
禽类的就巢性是一种由多基因控制的低遗传力性状,主要受环境、内分泌和遗传等因素的影响,其中遗传因素是决定性因素。繁殖激素是诱导就巢行为产生的直接原因,其中PRL是调控就巢行为产生和维持的主要因素,因此PRL和PRLR是繁殖相关激素基因中调控就巢性的主要候选基因,FSHβ、FSHR、LH、LHR、ESR、VIP和DRD1/2等基因通过调控PRL和PRLR基因的表达来调控禽类就巢行为的产生和维持。转录组学的研究同样也证实了HPG轴相关激素及其基因在调控就巢行为中的重要作用;通过相关的转录组学研究筛选出了大量的与繁殖和就巢性相关的蛋白编码基因和miRNAs,并初步揭示了调控就巢行为的生物学通路,如MAPK信号传导、TGF-β信号传导、催产素信号传导、GnRH信号传导和雌激素信号传导等。虽然近几年在转录组水平上筛选出了大量的与就巢性相关的候选基因和通路,但这些候选基因之间的互作关系、miRNA对基因的调控方式以及调控就巢性状的具体分子机制还不清楚,关于禽类就巢性的基因组学和蛋白质组学的研究也未见报道。此外,目前关于禽类就巢性的转录组学研究主要局限在卵巢、卵泡和下丘脑这3种组织中,缺少对于PRL合成和释放的垂体组织的研究。因此,在以后的研究中需要对调控禽类繁殖的HPG轴上的相关组织器官进行全面的分析,利用体外和活体实验对筛选出来的新编码基因和非编码基因进行具体功能的探索,同时利用全基因组、转录组和蛋白质组等多组学联合分析以及高通量染色体构象捕获(high-througnput chromosome conformation capture, Hi-C)等新技术来进一步揭示调控就巢行为的具体分子机制。(责任编委: 李辉)
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URL [本文引用: 1]
研究通过诱导就巢,观察并统计了东乡绿壳蛋鸡47～66周龄就巢发生情况。结果表明,观察期内平养绿壳蛋鸡群体平均卧息时间为10.5天(0～81天),除了产蛋外,27.7%的母鸡没有发生卧息行为;鸡群平均典型就巢次数为0.43次,群体平均就巢时间为12.3天(0～96天);超过30%的母鸡表现出典型就巢行为,其平均就巢持续时间约为29天。
URL [本文引用: 1]
研究通过诱导就巢,观察并统计了东乡绿壳蛋鸡47～66周龄就巢发生情况。结果表明,观察期内平养绿壳蛋鸡群体平均卧息时间为10.5天(0～81天),除了产蛋外,27.7%的母鸡没有发生卧息行为;鸡群平均典型就巢次数为0.43次,群体平均就巢时间为12.3天(0～96天);超过30%的母鸡表现出典型就巢行为,其平均就巢持续时间约为29天。
URLPMID:15971519 [本文引用: 3]
Prolactin (PRL) is generally accepted as crucial to the onset and maintenance of broodiness in avian species. The prolactin receptor (PRLR) plays an important role in the PRL signal transduction cascade. Two candidate genes, PRL and PRLR, were screened for polymorphisms in the chicken, and their genetic effects on broodiness were evaluated. Pedigreed hens (n = 155) of the Blue-shell chicken, a Chinese local breed, were observed for phenotypic broody traits including nesting days, broody days, repeats of broody cycles, and duration of broodiness. For polymorphism analysis, White Leg-horns, Hy-Line brown egg layers, Avian broilers, and some other Chinese local breeds were included. Fifteen sets of primers were used to amplify the nucleotide sequences of the promotor of PRL and exons of PRLR. The PCR products were screened for polymorphisms using single-stranded conformational polymorphism protocol. Sequencing revealed a 24-bp insertion occurring in the promotor, -377 090304 -354, of PRL (GenBank accession no. AB011434). A single nucleotide polymorphism (SNP), A9026G (GenBank accession no. AY237377), in exon 3 of PRLR was also detected, which led to a nucleotide transition in the 50905-untranslated region (50905-UTR) of PRLR cDNA. Two SNP, T14771C and G14820A (GenBank accession no. AY237376), were detected in exon 6 of the PRLR. The T14771C transition led to an amino acid variation, Leu340Ser, in PRLR, whereas the G14820A transition was a synonymous mutation. An association analysis showed that the genetic polymorphisms at PRLR3 and PRLR6 were not related to broodiness (P > 0.05), whereas the individuals without the insertion sequence at PRLpro2 were associated with broody traits (P 30%) of typical broody of genotypes +/- and -/- was higher (P < 0.01) than that of +/+. In addition, all White Leghorns were +/+ for PRLpro2, whereas local breeds with very strong broodiness were nearly all -/-. Homozygous insertion of the 24-bp sequence in the PRL promoter may decrease the expression of PRL, leading to nonbroodiness. The results suggested that PRLpro2 could be a genetic marker in breeding against broodiness in chickens.
URLMagsci [本文引用: 1]
<br><br><br><br><br>选择繁殖性能具有明显差异的4个鸡品种（莱航鸡、阳山鸡、丝羽乌骨鸡和隐性白洛克鸡）构建品种DNA池，采用测序的方法快速筛查鸡催乳素基因（chicken prolactin，cPRL）5′侧翼调控区、外显子区和部分内含子区约4500 bp范围内可能与产蛋性能相关的序列多态，共检测到13个SNPs和两个短片段（24 bp和15 bp）插入/缺失多态，其中在5′侧翼序列筛查到9个SNPs及两个短片段插入/缺失多态，在第2外显子筛查到1个SNP，在第5外显子筛查到两个SNPs，在第2内含子筛查到1个SNP；进一步利用生物信息学分析cPRL基因的5′侧翼调控序列，发现24 bp短片段的插入使莱航鸡比阳山鸡多出了1个Evi-1可能的结合位点（93分），C-2402T的变异则使阳山鸡比莱航鸡多出了1个C/EBPbeta可能的结合位点（94分），这两个结合位点是否影响cPRL基因的表达，影响鸡的就巢性和产蛋性能，还需要进一步研究。Abstract：Four chicken breeds (White Leghorn, Yangshan, Taihe Silkies, White Recessive Rocks) with different reproduction were applied to screen potential SNPs related to laying performance in the 5′flanking region, exon region and partial intron region of chicken prolactin (cPRL) gene. Totally almost 4500 bp were screened rapidly based on DNA pooling and sequencing, and thirteen single nucleotide polymorphisms (SNPs) and two indels (24 bp and 15 bp) were found, including nine SNPs and two indels in the 5′flanking region, one SNP in Exon 2, two SNPs in Exon 5 and one SNP in Intron 2 respectively. Furthermore, 5′flanking region of cPRL gene was analyzed by the website of http://motif.genome.ad.jp/. A possible Evi-1 binding site (score 93) was found in White Leghorn cPRL gene because of the 24 bp insertion, another possible C/EBPbeta binding site (score 94) was found in Yangshan cPRL gene because of the variation of C-2402T. Further studies need to be carried out to verify their effects on the expression of cPRL gene, the broodiness and laying performance of chickens.<br>
URLMagsci [本文引用: 1]
<br><br><br><br><br>选择繁殖性能具有明显差异的4个鸡品种（莱航鸡、阳山鸡、丝羽乌骨鸡和隐性白洛克鸡）构建品种DNA池，采用测序的方法快速筛查鸡催乳素基因（chicken prolactin，cPRL）5′侧翼调控区、外显子区和部分内含子区约4500 bp范围内可能与产蛋性能相关的序列多态，共检测到13个SNPs和两个短片段（24 bp和15 bp）插入/缺失多态，其中在5′侧翼序列筛查到9个SNPs及两个短片段插入/缺失多态，在第2外显子筛查到1个SNP，在第5外显子筛查到两个SNPs，在第2内含子筛查到1个SNP；进一步利用生物信息学分析cPRL基因的5′侧翼调控序列，发现24 bp短片段的插入使莱航鸡比阳山鸡多出了1个Evi-1可能的结合位点（93分），C-2402T的变异则使阳山鸡比莱航鸡多出了1个C/EBPbeta可能的结合位点（94分），这两个结合位点是否影响cPRL基因的表达，影响鸡的就巢性和产蛋性能，还需要进一步研究。Abstract：Four chicken breeds (White Leghorn, Yangshan, Taihe Silkies, White Recessive Rocks) with different reproduction were applied to screen potential SNPs related to laying performance in the 5′flanking region, exon region and partial intron region of chicken prolactin (cPRL) gene. Totally almost 4500 bp were screened rapidly based on DNA pooling and sequencing, and thirteen single nucleotide polymorphisms (SNPs) and two indels (24 bp and 15 bp) were found, including nine SNPs and two indels in the 5′flanking region, one SNP in Exon 2, two SNPs in Exon 5 and one SNP in Intron 2 respectively. Furthermore, 5′flanking region of cPRL gene was analyzed by the website of http://motif.genome.ad.jp/. A possible Evi-1 binding site (score 93) was found in White Leghorn cPRL gene because of the 24 bp insertion, another possible C/EBPbeta binding site (score 94) was found in Yangshan cPRL gene because of the variation of C-2402T. Further studies need to be carried out to verify their effects on the expression of cPRL gene, the broodiness and laying performance of chickens.<br>
[D].
URL [本文引用: 1]
鸟类的排卵过程为卵巢发育成熟并产生成熟的卵泡,卵泡脱离卵巢通过输卵管的各个部分形成完整的蛋,并最终排出。而在这个过程中卵巢和输卵管受到诸多的生长因子和激素的调控。通过选育培养出的蛋鸡在进入高产期后几乎每天产蛋,其原因在于蛋鸡卵泡在生殖阶段可持续发育、并排卵和受精,卵巢排卵后亦不形成黄体,生殖周期极短,是作为生殖系统研究的理想试验动物。本试验以中国地方特色蛋鸡品种绿壳蛋鸡为试验材料,测定不同产蛋时期的等级前卵泡、优势等级卵泡和输卵管中的神经生长因子NGF及其受体的mRNA表达量,并对产蛋期和就巢期的组织进行NGF及其受体的表达对比,同时通过体外培养的方式对绿壳蛋鸡的F1卵泡颗粒细胞培养并添加NGF蛋白,测定各个受体的mRNA表达量和性腺激素的分泌量。结果如下：通过对绿壳蛋鸡不同产蛋阶段(19W、33W、50W、64W)的生殖组织进行采样并提取mRNA,并进行荧光定量对其卵泡和输卵管的NGF及其受体的mRNA表达量测定。结果表明：NGF在绿壳蛋鸡各个产蛋时期中的卵泡和输卵管中都有表达；在等级前卵泡中,除了产蛋末期外,其他时期的小白卵泡SWF的NGF和其高亲和力受体酪氨酸激酶A (TrkA)的mRNA表达量要显著高于其他等级前卵泡(P0.05),且随着等级前卵泡的发育,NGF的表达量呈现下降的趋势,而在整个产蛋时期中,NGF在产蛋高峰期表达量最高；在等级卵泡中,NGF及其受体在F4卵泡中表达量在各时期都较高,且在产蛋前期和高峰期,F1-F4的mRNA表达量都高于F5的mRNA表达量,而在整个产蛋周期中,NGF的表达量在产蛋高峰期处于峰值；每个时期的输卵管各部位都有NGF的表达,且表达量随着绿壳蛋鸡产蛋周期的变化而变化,在产蛋后期NGF及其高亲和力受体表达量最高。以上结果说明,NGF的表达量与蛋鸡的生殖状况和产蛋性能相关,推测NGF可能参与了调控卵巢和输卵管生长发育并有助于蛋鸡产蛋。利用荧光定量测定方法对绿壳蛋鸡产蛋期和就巢期各组织的NGF及其受体的表达进行研究。结果表明：产蛋期的卵巢大于就巢期卵巢,且具有小黄卵泡SYF和F1-F6卵泡,而就巢期卵巢处于萎缩形态,没有优势卵泡；在产蛋期的卵巢基质中,NGF及其受体显著高于就巢期(P0.05)；在等级前卵泡中,SWF和LWF中的NGF及其高亲和力受体在产蛋期的mRNA的表达量显著高于就巢期(P0.05)。以上结果说明NGF及其受体在卵巢组织中的表达与禽类产蛋周期有影响,并可能参与了禽类产蛋时期与就巢时期的调控,NGF的表达有助于激活非发情期卵巢并改变卵巢形态,促使等级前卵泡发育成为等级优势卵泡。通过对F1卵泡颗粒细胞离体培养,添加NGF蛋白并进行受体基因的荧光定量及激素的Elisa检查,探究NGF蛋白在蛋鸡卵泡发育过程中的作用。结果表明：在添加不同浓度NGF蛋白处理后,颗粒细胞中的p75受体的表达量显著升高(P0.05),且随着NGF蛋白的浓度升高而升高；处理不同浓度的NGF蛋白后,颗粒细胞中的FSHR和LHR也显著升高(P0.05),且存在正相关关系；通过Elisa检查发现,添加NGF蛋白处理后的颗粒细胞中雌激素的分泌显著升高(P＜0.05),而孕激素则相反;在依次添加NGF和FSH处理后发现,雌激素的分泌在单独添加NGF和FSH时都将显著升高(P0.05),而且同时添加有叠加效果,而孕激素的分泌则相反。以上结果表明,NGF在禽类卵泡中能促进其LHR和FSHR的表达,通过加强细胞对激素的敏感性来调控卵泡的发育和排卵,并且影响动物的生理周期。综上所述,NGF及其受体能促进禽类早期卵巢和输卵管的生长和发育,并在卵巢和输卵管成熟后,促进卵巢卵泡的发育以及排卵,同时在也影响鸡的生殖周期。
URL [本文引用: 1]
鸟类的排卵过程为卵巢发育成熟并产生成熟的卵泡,卵泡脱离卵巢通过输卵管的各个部分形成完整的蛋,并最终排出。而在这个过程中卵巢和输卵管受到诸多的生长因子和激素的调控。通过选育培养出的蛋鸡在进入高产期后几乎每天产蛋,其原因在于蛋鸡卵泡在生殖阶段可持续发育、并排卵和受精,卵巢排卵后亦不形成黄体,生殖周期极短,是作为生殖系统研究的理想试验动物。本试验以中国地方特色蛋鸡品种绿壳蛋鸡为试验材料,测定不同产蛋时期的等级前卵泡、优势等级卵泡和输卵管中的神经生长因子NGF及其受体的mRNA表达量,并对产蛋期和就巢期的组织进行NGF及其受体的表达对比,同时通过体外培养的方式对绿壳蛋鸡的F1卵泡颗粒细胞培养并添加NGF蛋白,测定各个受体的mRNA表达量和性腺激素的分泌量。结果如下：通过对绿壳蛋鸡不同产蛋阶段(19W、33W、50W、64W)的生殖组织进行采样并提取mRNA,并进行荧光定量对其卵泡和输卵管的NGF及其受体的mRNA表达量测定。结果表明：NGF在绿壳蛋鸡各个产蛋时期中的卵泡和输卵管中都有表达；在等级前卵泡中,除了产蛋末期外,其他时期的小白卵泡SWF的NGF和其高亲和力受体酪氨酸激酶A (TrkA)的mRNA表达量要显著高于其他等级前卵泡(P0.05),且随着等级前卵泡的发育,NGF的表达量呈现下降的趋势,而在整个产蛋时期中,NGF在产蛋高峰期表达量最高；在等级卵泡中,NGF及其受体在F4卵泡中表达量在各时期都较高,且在产蛋前期和高峰期,F1-F4的mRNA表达量都高于F5的mRNA表达量,而在整个产蛋周期中,NGF的表达量在产蛋高峰期处于峰值；每个时期的输卵管各部位都有NGF的表达,且表达量随着绿壳蛋鸡产蛋周期的变化而变化,在产蛋后期NGF及其高亲和力受体表达量最高。以上结果说明,NGF的表达量与蛋鸡的生殖状况和产蛋性能相关,推测NGF可能参与了调控卵巢和输卵管生长发育并有助于蛋鸡产蛋。利用荧光定量测定方法对绿壳蛋鸡产蛋期和就巢期各组织的NGF及其受体的表达进行研究。结果表明：产蛋期的卵巢大于就巢期卵巢,且具有小黄卵泡SYF和F1-F6卵泡,而就巢期卵巢处于萎缩形态,没有优势卵泡；在产蛋期的卵巢基质中,NGF及其受体显著高于就巢期(P0.05)；在等级前卵泡中,SWF和LWF中的NGF及其高亲和力受体在产蛋期的mRNA的表达量显著高于就巢期(P0.05)。以上结果说明NGF及其受体在卵巢组织中的表达与禽类产蛋周期有影响,并可能参与了禽类产蛋时期与就巢时期的调控,NGF的表达有助于激活非发情期卵巢并改变卵巢形态,促使等级前卵泡发育成为等级优势卵泡。通过对F1卵泡颗粒细胞离体培养,添加NGF蛋白并进行受体基因的荧光定量及激素的Elisa检查,探究NGF蛋白在蛋鸡卵泡发育过程中的作用。结果表明：在添加不同浓度NGF蛋白处理后,颗粒细胞中的p75受体的表达量显著升高(P0.05),且随着NGF蛋白的浓度升高而升高；处理不同浓度的NGF蛋白后,颗粒细胞中的FSHR和LHR也显著升高(P0.05),且存在正相关关系；通过Elisa检查发现,添加NGF蛋白处理后的颗粒细胞中雌激素的分泌显著升高(P＜0.05),而孕激素则相反;在依次添加NGF和FSH处理后发现,雌激素的分泌在单独添加NGF和FSH时都将显著升高(P0.05),而且同时添加有叠加效果,而孕激素的分泌则相反。以上结果表明,NGF在禽类卵泡中能促进其LHR和FSHR的表达,通过加强细胞对激素的敏感性来调控卵泡的发育和排卵,并且影响动物的生理周期。综上所述,NGF及其受体能促进禽类早期卵巢和输卵管的生长和发育,并在卵巢和输卵管成熟后,促进卵巢卵泡的发育以及排卵,同时在也影响鸡的生殖周期。
URL [本文引用: 4]
为探究新疆伊犁鹅繁殖周期中血浆主要生殖激素、卵巢形态及卵泡发育的变化规律,试验以健康状况良好、体重相近的2岁伊犁鹅为试验对象,分别在产蛋期、就巢期及休产期各随机选取32只,采集1次血样,测定激素水平;在各时期分别选取5只采集卵巢组织,观测卵巢及卵泡发育情况。结果表明,在产蛋期,伊犁鹅的GnRH分别极显著高于就巢期和休产期16.78%和58.29%(P0.01);PRL显著低于就巢期13.00%(P0.05),极显著高于休产期28.94%(P0.01);FSH高于就巢期6.98%(P0.05),极显著高于休产期21.12%(P0.01);LH极显著低于就巢期8.38%(P0.01),极显著高于休产期16.84%(P0.01);E2水平分别极显著高于就巢期和休产期29.80%和112.40%(P0.01);P4水平分别极显著高于就巢期和休产期28.89%和30.34%(P0.01)。产蛋期伊犁鹅的卵巢体积和重量均极显著大于就巢期和休产期(P0.01)。产蛋期伊犁鹅成熟卵泡与就巢期和休产期相比明显较大,次级卵泡数量较多,初级卵泡数量较少;就巢期卵巢上生长及成熟卵泡发生闭锁,颗粒胞层收缩,向内凹陷,胞质出现空腔,各级卵泡出现萎缩;与就巢期相比,休产期卵巢上卵泡胞质空腔变大,向内凹陷程度进一步加深,大量原始卵泡均匀排列。由此可见,在产蛋期,伊犁鹅的GnRH、E2、LH反应活跃,对卵巢卵泡发育起主导作用;在就巢期,PRL、LH维持较高的分泌水平,协同维持就巢。
URL [本文引用: 4]
为探究新疆伊犁鹅繁殖周期中血浆主要生殖激素、卵巢形态及卵泡发育的变化规律,试验以健康状况良好、体重相近的2岁伊犁鹅为试验对象,分别在产蛋期、就巢期及休产期各随机选取32只,采集1次血样,测定激素水平;在各时期分别选取5只采集卵巢组织,观测卵巢及卵泡发育情况。结果表明,在产蛋期,伊犁鹅的GnRH分别极显著高于就巢期和休产期16.78%和58.29%(P0.01);PRL显著低于就巢期13.00%(P0.05),极显著高于休产期28.94%(P0.01);FSH高于就巢期6.98%(P0.05),极显著高于休产期21.12%(P0.01);LH极显著低于就巢期8.38%(P0.01),极显著高于休产期16.84%(P0.01);E2水平分别极显著高于就巢期和休产期29.80%和112.40%(P0.01);P4水平分别极显著高于就巢期和休产期28.89%和30.34%(P0.01)。产蛋期伊犁鹅的卵巢体积和重量均极显著大于就巢期和休产期(P0.01)。产蛋期伊犁鹅成熟卵泡与就巢期和休产期相比明显较大,次级卵泡数量较多,初级卵泡数量较少;就巢期卵巢上生长及成熟卵泡发生闭锁,颗粒胞层收缩,向内凹陷,胞质出现空腔,各级卵泡出现萎缩;与就巢期相比,休产期卵巢上卵泡胞质空腔变大,向内凹陷程度进一步加深,大量原始卵泡均匀排列。由此可见,在产蛋期,伊犁鹅的GnRH、E2、LH反应活跃,对卵巢卵泡发育起主导作用;在就巢期,PRL、LH维持较高的分泌水平,协同维持就巢。
[D].
[本文引用: 1]
[本文引用: 1]
URLPMID:29739971 [本文引用: 24]
Abstract Broodiness in laying hens results in atrophy of the ovary and consequently decreases productivity. However, the regulatory mechanisms that drive ovary development remain elusive. Thus, we collected atrophic ovaries (AO) from 380-day-old broody chickens (BC) and normal ovaries (NO) from even-aged egg-laying hens (EH) for RNA sequencing. We identified 3,480 protein-coding transcripts that were differentially expressed (DE), including 1,719 that were down-regulated and 1,761 that were up-regulated in AO. There were 959 lncRNA transcripts that were DE, including 56 that were down-regulated and 903 that were up-regulated. Among the116 miRNAs that were DE, 79 were down-regulated and 37 were up-regulated in AO. Numerous DE protein-coding transcripts and target genes for miRNAs/lncRNAs were significantly enriched in reproductive processes, cell proliferation, and apoptosis pathways. A miRNA-intersection gene-pathway network was constructed by considering target relationships and correlation of the expression levels between ovary development-related genes and miRNAs. We also constructed a competing endogenous RNA (ceRNA) network by integrating competing relationships between protein-coding genes and lncRNA transcripts, and identified several lncRNA transcripts predicted to regulate the CASP6, CYP1B1, GADD45, MMP2, and SMAS2 genes. In conclusion, we discovered protein-coding genes, miRNAs, and lncRNA transcripts that are candidate regulators of ovary development in broody chickens.
[D].
URL [本文引用: 2]
本研究由番鸭就巢规律、番鸭就巢期生殖激素的变化规律和番鸭催乳素基因的微卫星多态性分析三个部分组成: 实验一采用“泄殖腔外托蛋法”对番鸭就巢规律跟踪测试,结果表明:(1) 番鸭个体间就巢期的差异较大,就巢期短则30天,长者达106天,且间歇休产频繁出现。第一、第二产蛋期就巢鸭分别占18.25%和17.78%,两者相差不大。(2) 番鸭开产体重与开产日龄呈显著正相关(P0.05)(R=0.198);开产体重与就巢期长短呈极显著正相关(P0.01)(R=0.598)。番鸭在就巢期间的体重变化与就巢时间长短呈极显著正相关(P0.01)(R=0.973)。(3) 产蛋量与就巢期长短呈极显著的负相关(P0.01)(R=-0.567),就巢母鸭比无就巢母鸭开产早,同时产蛋高峰也来得早,但产蛋高峰持续时间较短,产蛋率下降快;而无就巢性的母鸭产蛋率较高,且产蛋高峰时间持续较长。 试验二采用放射免疫分析法对番鸭就巢期生殖激素的变化规律进行测试,结果表明:(1) 就巢期血浆催乳素(PRL)的浓度水平(0.68±0.11ng/ml)极显著高于醒抱期浓度水平(0.33±0.04ng/ml)(P0.01);(2) 就巢期血浆血管活性肠肽(VIP)的浓度水平(587.61±119.17pg/mL)极显著高于醒抱期浓度水平(307.44±96.92pg/mL)(P0.01);(3) 醒抱前后血浆促黄体素(LH)浓度水平差异很大,醒抱期促黄体素浓度水平(33.17±15.49mIU/ml)极显著高于就巢期浓度水平(5.56±1.99mIU/ml)(P0.01);(4) 醒抱期血浆中雌激素(E_2)浓度水平(1.9±1.17ng/ml)极显著高于就巢期浓度水平(0.9±0.65ng/ml)(P0.01):(5) 醒抱期血清孕酮(PROG)的浓度水平(15.56±4.00ng/mL)极显著高于就巢期浓度水平(6.65±2.04ng/mL)(P0.01);(6) 醒抱前后,PRL与VIP浓度水平变化趋势基本一致,呈极显著的正相关(R_(PRL.VIP)=0.857);(7) 醒抱前后,E_2、LH和PROG浓度水平变化趋势基本一致,相互之间呈显著的正相关(R_(E2.LH)=0.641,R_(E2.PROG)=0.817,R_(LH.PROG)=0.746);(8) 就巢期和醒抱期PRL、VIP和E_2、LH、PROG浓度水平变化趋势正好相反,呈显著的负相关(R_(PRL.E2)=-0.666,R_(PRL.LH)=-0.923,R_(PRL.PROG)=-0.718,R_(VIP.E2)=-0.846,R_(VIP.LH)=-0.726,R_(VIP.PROG)=-0.806)。 根据番鸭就巢期生殖激素的变化规律,可以推测番鸭就巢的内分泌机理:番鸭产蛋期在促黄体素(LH)的作用下,卵泡分泌大量雌激素(E_2)和孕酮(PROG),这两个性腺激素促进下丘脑合成并释放血管活性肠肽(VIP),后者促进垂体前叶分泌催乳素(PRL)。高浓度水平催乳素使母鸭发生就巢行为,同时抑制促黄体素(LH)的分泌和直接抑制卵巢活动,使卵巢萎缩产蛋停止;并且也间接抑制了雌激素(E_2)和孕酮(PROG)的分泌。
URL [本文引用: 2]
本研究由番鸭就巢规律、番鸭就巢期生殖激素的变化规律和番鸭催乳素基因的微卫星多态性分析三个部分组成: 实验一采用“泄殖腔外托蛋法”对番鸭就巢规律跟踪测试,结果表明:(1) 番鸭个体间就巢期的差异较大,就巢期短则30天,长者达106天,且间歇休产频繁出现。第一、第二产蛋期就巢鸭分别占18.25%和17.78%,两者相差不大。(2) 番鸭开产体重与开产日龄呈显著正相关(P0.05)(R=0.198);开产体重与就巢期长短呈极显著正相关(P0.01)(R=0.598)。番鸭在就巢期间的体重变化与就巢时间长短呈极显著正相关(P0.01)(R=0.973)。(3) 产蛋量与就巢期长短呈极显著的负相关(P0.01)(R=-0.567),就巢母鸭比无就巢母鸭开产早,同时产蛋高峰也来得早,但产蛋高峰持续时间较短,产蛋率下降快;而无就巢性的母鸭产蛋率较高,且产蛋高峰时间持续较长。 试验二采用放射免疫分析法对番鸭就巢期生殖激素的变化规律进行测试,结果表明:(1) 就巢期血浆催乳素(PRL)的浓度水平(0.68±0.11ng/ml)极显著高于醒抱期浓度水平(0.33±0.04ng/ml)(P0.01);(2) 就巢期血浆血管活性肠肽(VIP)的浓度水平(587.61±119.17pg/mL)极显著高于醒抱期浓度水平(307.44±96.92pg/mL)(P0.01);(3) 醒抱前后血浆促黄体素(LH)浓度水平差异很大,醒抱期促黄体素浓度水平(33.17±15.49mIU/ml)极显著高于就巢期浓度水平(5.56±1.99mIU/ml)(P0.01);(4) 醒抱期血浆中雌激素(E_2)浓度水平(1.9±1.17ng/ml)极显著高于就巢期浓度水平(0.9±0.65ng/ml)(P0.01):(5) 醒抱期血清孕酮(PROG)的浓度水平(15.56±4.00ng/mL)极显著高于就巢期浓度水平(6.65±2.04ng/mL)(P0.01);(6) 醒抱前后,PRL与VIP浓度水平变化趋势基本一致,呈极显著的正相关(R_(PRL.VIP)=0.857);(7) 醒抱前后,E_2、LH和PROG浓度水平变化趋势基本一致,相互之间呈显著的正相关(R_(E2.LH)=0.641,R_(E2.PROG)=0.817,R_(LH.PROG)=0.746);(8) 就巢期和醒抱期PRL、VIP和E_2、LH、PROG浓度水平变化趋势正好相反,呈显著的负相关(R_(PRL.E2)=-0.666,R_(PRL.LH)=-0.923,R_(PRL.PROG)=-0.718,R_(VIP.E2)=-0.846,R_(VIP.LH)=-0.726,R_(VIP.PROG)=-0.806)。 根据番鸭就巢期生殖激素的变化规律,可以推测番鸭就巢的内分泌机理:番鸭产蛋期在促黄体素(LH)的作用下,卵泡分泌大量雌激素(E_2)和孕酮(PROG),这两个性腺激素促进下丘脑合成并释放血管活性肠肽(VIP),后者促进垂体前叶分泌催乳素(PRL)。高浓度水平催乳素使母鸭发生就巢行为,同时抑制促黄体素(LH)的分泌和直接抑制卵巢活动,使卵巢萎缩产蛋停止;并且也间接抑制了雌激素(E_2)和孕酮(PROG)的分泌。
URLPMID:9495498 [本文引用: 2]
Plasma prolactin (PRL) levels rise following long daily photostimulation and increase dramatically at the onset of incubation behavior. Previous work has shown that a daily rhythm in PRL secretion may occur, with the lowest PRL levels found prior to lights out and the highest levels found prior to lights on. It has been suggested that an early event in the onset of incubation behavior may be an increase in nocturnal PRL levels. A retrospective study was conducted to contrast the morning and evening PRL secretion patterns at weekly intervals throughout the reproductive cycle in birds that exhibited: 1) incubation behavior; 2) high egg production and low nesting frequency for the last 16 wk of a 21-wk reproductive cycle; 3) high egg production while nesting frequently; or 4) photorefractoriness (defined by a cessation of egg production without incubation behavior). All hens showed an increase in PRL levels following photostimulation. When day and night PRL levels for each hen were compared over the entire reproductive cycle, more than 50% of those studied had significantly higher PRL levels at the end of the scotophase than at the end of the photophase. Circulating PRL levels increased greatly with the onset of incubation behavior, but morning and evening PRL levels changed in parallel. Good layers had moderate PRL levels throughout egg production, but PRL levels did not differ among laying hens with high or low nesting frequency. Plasma PRL levels declined to low levels in photorefractory hens. These results show that daytime PRL measurements accurately reflect reproductive state, and that moderate PRL levels seem consistent with optimum egg production.
URL [本文引用: 3]
在现代高效的养禽业中,就巢性已成为影响家禽繁殖力的重要因素.本文对以催乳素、促性腺激素释放激素、促卵泡生成激素、促黄体生成素、雌激素、孕酮、血管活性肠肽、多巴胺和5-羟色胺等调控禽类就巢的研究进行综述.
URL [本文引用: 3]
在现代高效的养禽业中,就巢性已成为影响家禽繁殖力的重要因素.本文对以催乳素、促性腺激素释放激素、促卵泡生成激素、促黄体生成素、雌激素、孕酮、血管活性肠肽、多巴胺和5-羟色胺等调控禽类就巢的研究进行综述.
URL [本文引用: 3]
为了研究定安鹅不同繁殖周期脑 下垂体激素与催乳素分泌水平的变化规律,试验采用酶联免疫吸附(ELISA)双抗体夹心法测定了定安鹅繁殖周期中开产期、产蛋期、就巢期血浆中促性腺激素 释放激素、促黄体素、促卵泡激素和催乳素浓度。结果表明:在定安鹅的繁殖周期中,促性腺激素释放激素、促黄体素、促卵泡激素和催乳素在不同时期均有不同变 化,对鹅产蛋、就巢、开产等繁殖行为具有十分重要的作用,催乳素对于引发、维持定安鹅的就巢行为具有决定性的作用,低水平的促黄体素与就巢行为有关,而就 巢期长短直接影响定安鹅的产蛋期和产蛋量。
URL [本文引用: 3]
为了研究定安鹅不同繁殖周期脑 下垂体激素与催乳素分泌水平的变化规律,试验采用酶联免疫吸附(ELISA)双抗体夹心法测定了定安鹅繁殖周期中开产期、产蛋期、就巢期血浆中促性腺激素 释放激素、促黄体素、促卵泡激素和催乳素浓度。结果表明:在定安鹅的繁殖周期中,促性腺激素释放激素、促黄体素、促卵泡激素和催乳素在不同时期均有不同变 化,对鹅产蛋、就巢、开产等繁殖行为具有十分重要的作用,催乳素对于引发、维持定安鹅的就巢行为具有决定性的作用,低水平的促黄体素与就巢行为有关,而就 巢期长短直接影响定安鹅的产蛋期和产蛋量。
URL [本文引用: 2]
为探讨产蛋期和就巢期峨眉黑鸡生理变化规律和就巢调控的内分泌机制,试验分别选取10只产蛋期和10只就巢期峨眉黑鸡作为研究对象,通过测定血清生化指标、矿物质元素以及内分泌激素水平,对比分析产蛋期与就巢期各指标的差异。结果显示:就巢期鸡血清中总胆固醇(TC)、总蛋白(TP)、白蛋白(Alb)以及矿物元素(Ca、P)含量显著低于产蛋期(P<0.05),甘油三酯(TG)和Fe含量极显著低于产蛋期(P<0.01),葡萄糖(Glu)含量显著高于产蛋期(P<0.05);就巢期免疫球蛋白IgM和IgG含量极显著低于产蛋组(P<0.01);内分泌激素方面,就巢期血清促卵泡激素(FSH)含量显著高于产蛋期(P<0.05),雌二醇(E_2)、促黄体系(LH)、促性腺激素释放激素(GnRH)、孕酮(P4)含量低于产蛋期(P>0.05),催乳素(PRL)、血管活性肠肽(VIP)含量高于产蛋期(P>0.05)。研究表明,鸡就巢期采食量抑制会导致相关营养性血清生化指标显著降低,机体免疫力下降。
URL [本文引用: 2]
为探讨产蛋期和就巢期峨眉黑鸡生理变化规律和就巢调控的内分泌机制,试验分别选取10只产蛋期和10只就巢期峨眉黑鸡作为研究对象,通过测定血清生化指标、矿物质元素以及内分泌激素水平,对比分析产蛋期与就巢期各指标的差异。结果显示:就巢期鸡血清中总胆固醇(TC)、总蛋白(TP)、白蛋白(Alb)以及矿物元素(Ca、P)含量显著低于产蛋期(P<0.05),甘油三酯(TG)和Fe含量极显著低于产蛋期(P<0.01),葡萄糖(Glu)含量显著高于产蛋期(P<0.05);就巢期免疫球蛋白IgM和IgG含量极显著低于产蛋组(P<0.01);内分泌激素方面,就巢期血清促卵泡激素(FSH)含量显著高于产蛋期(P<0.05),雌二醇(E_2)、促黄体系(LH)、促性腺激素释放激素(GnRH)、孕酮(P4)含量低于产蛋期(P>0.05),催乳素(PRL)、血管活性肠肽(VIP)含量高于产蛋期(P>0.05)。研究表明,鸡就巢期采食量抑制会导致相关营养性血清生化指标显著降低,机体免疫力下降。
URLMagsci [本文引用: 1]
采用多巴胺(dopamine,DA)和5-羟色胺(5- hydroxy tryptamine,5-HT)的拮抗剂奥氮平(olanzapine,Ola)作为调控物,研究奥氮平对美国王鸽产蛋性能的影响并探讨其作用机制.选 720日龄美国王鸽120对,随机分成4组;对照组饲喂基础日粮,试验组分别添加0.25、0.75和1.25 mg·kg-1臭氮平,试验期60 d.试验结果显示:1)与对照组相比,0.75和1.25 mg·kg-1奥氮平组月产蛋个数分别提高了20.24%(P＜0.05)和23.41%(P＜0.05),产蛋间隔分别缩短了29.83% (P＜0.05)和37.46%(P＜0.05),血清总蛋白水平分别提高了7.33%(P＜0.05)和11.59%(P＜0.05),血清尿素氮水平 分别降低了32.6%(P＜0.05)和47.8%(P＜0.05);2)各组受精率和孵化率差异无统计学意义(P＞0.05);0.75和1.25 mg·kg-1奥氮平组血清催乳素水平较对照组分别降低了21.90%(P＜0.05)和34.53%(P＜0.05),促卵泡生成素水平分别提高了 24.0%(P＜0.05)和28.5%(P＜0.05),血清孕酮水平分别升高了33.7%(P＜0.05)和42.0%(P＜0.05).表明DA和 5-HT的受体拮抗剂奥氮平可以降低血清催乳素(PRL)的分泌,提高促卵泡刺激素和孕酮的分泌,对禽类的就巢发生和维持具有抑制作用;也说明DA和5- HT对禽类就巢的发生和PRL的分泌具有促进作用.
URLMagsci [本文引用: 1]
采用多巴胺(dopamine,DA)和5-羟色胺(5- hydroxy tryptamine,5-HT)的拮抗剂奥氮平(olanzapine,Ola)作为调控物,研究奥氮平对美国王鸽产蛋性能的影响并探讨其作用机制.选 720日龄美国王鸽120对,随机分成4组;对照组饲喂基础日粮,试验组分别添加0.25、0.75和1.25 mg·kg-1臭氮平,试验期60 d.试验结果显示:1)与对照组相比,0.75和1.25 mg·kg-1奥氮平组月产蛋个数分别提高了20.24%(P＜0.05)和23.41%(P＜0.05),产蛋间隔分别缩短了29.83% (P＜0.05)和37.46%(P＜0.05),血清总蛋白水平分别提高了7.33%(P＜0.05)和11.59%(P＜0.05),血清尿素氮水平 分别降低了32.6%(P＜0.05)和47.8%(P＜0.05);2)各组受精率和孵化率差异无统计学意义(P＞0.05);0.75和1.25 mg·kg-1奥氮平组血清催乳素水平较对照组分别降低了21.90%(P＜0.05)和34.53%(P＜0.05),促卵泡生成素水平分别提高了 24.0%(P＜0.05)和28.5%(P＜0.05),血清孕酮水平分别升高了33.7%(P＜0.05)和42.0%(P＜0.05).表明DA和 5-HT的受体拮抗剂奥氮平可以降低血清催乳素(PRL)的分泌,提高促卵泡刺激素和孕酮的分泌,对禽类的就巢发生和维持具有抑制作用;也说明DA和5- HT对禽类就巢的发生和PRL的分泌具有促进作用.
URL [本文引用: 1]
本试验利用多巴胺(dopamine,DA)和5-羟色胺(5-hydroxytryptamine,5-HT)受体阻断剂作用于皖西白鹅的就巢开始,进行醒抱和就巢的内分泌调控试验。于就巢第1d开始用药(氯丙嗪:80mg/kg·d,赛更啶:8mg/kg·d),连用3d。醒抱试验结果为:氯丙嗪处理组的平均就巢天数为2.5±0.8d,赛更啶处理组为3.8±1.2d,氯丙嗪和赛更啶共同处理组为1.0±0.2d,均极显著低于对照组(35d,P0.01)。就巢的内分泌调控试验结果为:用药物共同处理后,试验组E2先升后降,并维持较高水平(256.74±23.98ng/ml),对照组下降较明显,并维持较低水平(162.65±15.36ng/ml);试验组催乳素(prolactin,PRL)由开始就巢时的0.26±0.06ng/ml降至0.18±0.02ng/ml,而对照组的PRL则从就巢开始的0.26±0.06ng/ml一直上升至0.38±0.06ng/ml;外周P4浓度从就巢开始时的46.18±3.36nmol/ml显著降至16.67±4.35nmol/ml(P0.05),并维持低水平;而FSH的变化则无显著差异。以上试验结果提示,DA和5-HT阻断剂可以降低禽类机体内PRL分泌,对禽类就巢的发生和维持具有抑制作用,同时表明DA和5-HT对于禽类就巢的发生和垂体PRL的释放具有一定的协同促进作用。
URL [本文引用: 1]
本试验利用多巴胺(dopamine,DA)和5-羟色胺(5-hydroxytryptamine,5-HT)受体阻断剂作用于皖西白鹅的就巢开始,进行醒抱和就巢的内分泌调控试验。于就巢第1d开始用药(氯丙嗪:80mg/kg·d,赛更啶:8mg/kg·d),连用3d。醒抱试验结果为:氯丙嗪处理组的平均就巢天数为2.5±0.8d,赛更啶处理组为3.8±1.2d,氯丙嗪和赛更啶共同处理组为1.0±0.2d,均极显著低于对照组(35d,P0.01)。就巢的内分泌调控试验结果为:用药物共同处理后,试验组E2先升后降,并维持较高水平(256.74±23.98ng/ml),对照组下降较明显,并维持较低水平(162.65±15.36ng/ml);试验组催乳素(prolactin,PRL)由开始就巢时的0.26±0.06ng/ml降至0.18±0.02ng/ml,而对照组的PRL则从就巢开始的0.26±0.06ng/ml一直上升至0.38±0.06ng/ml;外周P4浓度从就巢开始时的46.18±3.36nmol/ml显著降至16.67±4.35nmol/ml(P0.05),并维持低水平;而FSH的变化则无显著差异。以上试验结果提示,DA和5-HT阻断剂可以降低禽类机体内PRL分泌,对禽类就巢的发生和维持具有抑制作用,同时表明DA和5-HT对于禽类就巢的发生和垂体PRL的释放具有一定的协同促进作用。
[D].
URL [本文引用: 2]
动物垂体前叶分泌的催乳素(Prolactin,PRL)具有广泛的生物学效应,在家畜中PRL主要涉及到繁殖性能,在家禽中PRL主要影响就巢行为,进而影响到产蛋性能等繁殖性状,这些作用均由催乳素受体(prolactin receptor,PRLR)调节。大量研究表明,催乳素(PRL)的生理作用是通过位于靶细胞膜上的催乳素受体(PRLR)介导的。在家禽中,PRLR基因是与就巢性密切相关的功能基因。本试验应用荧光定量PCR技术检测了皖西白鹅在产前一周、就巢期、产蛋后十天及休产期时PRLR基因在垂体、下丘脑、卵巢三个组织中的表达水平,探讨了三个组织中PRLR基因在不同繁殖时期mRNA的表达规律。 研究发现PRLR基因在下丘脑、垂体、卵巢上均有表达,表达量从高到低依次为:卵巢下丘脑垂体,各组织间PRLR mRNA的表达量存在明显差异,其中卵巢和下丘脑上的表达量差异显著(P0.05),卵巢和垂体上的表达量差异极显著(P0.01)。对不同时期分析结果显示PRLR mRNA表达量从高到低依次是:就巢期产蛋前一周产蛋后十天休产期。其中就巢期PRLR基因的表达量分别是产蛋前一周、产蛋后十天、休产期的4倍、40倍、45倍,差异均极显著(P0.01)。对同一组织不同时期PRLR mRNA表达量分析表明,垂体四个时期间PRLR基因表达量从高到低依次是:就巢期休产期产蛋后十天产蛋前一周,就巢期的PRLR基因的表达量极显著高于其它三个时期(P0.01),其它三个时期之间差异不显著(P0.05);下丘脑四个时期间PRLR基因表达量从高到低依次是:就巢期产蛋后十天产蛋前一周休产期,就巢期和其它三个时期的PRLR基因的表达量差异均极显著(P0.01),其它三个时期之间差异不显著(P0.05);卵巢四个时期PRLR基因的表达量从高到低依次是:就巢期产蛋前一周休产期产蛋后十天,就巢期的PRLR基因的表达量极显著高于其它三个时期(P0.01),产蛋前一周的PRLR基因的表达量高于产蛋后十天和休产期,差异极显著(P0.01)。 在就巢期,三个组织PRLR基因的表达量都很高,之间差异不显著(P0.05)。其中垂体就巢期三个时间段的PRLR基因表达量从高到低依次是:就巢30-31天就巢1-3天就巢14-16天,就巢30-31天PRLR基因的表达量是就巢14-16天的3倍,差异显著(P0.05);下丘脑和卵巢就巢期三个时间段PRLR基因表达量从高到低依次是:就巢1-3天就巢14-16天就巢30-31天,差异不显著(P0.05)。
URL [本文引用: 2]
动物垂体前叶分泌的催乳素(Prolactin,PRL)具有广泛的生物学效应,在家畜中PRL主要涉及到繁殖性能,在家禽中PRL主要影响就巢行为,进而影响到产蛋性能等繁殖性状,这些作用均由催乳素受体(prolactin receptor,PRLR)调节。大量研究表明,催乳素(PRL)的生理作用是通过位于靶细胞膜上的催乳素受体(PRLR)介导的。在家禽中,PRLR基因是与就巢性密切相关的功能基因。本试验应用荧光定量PCR技术检测了皖西白鹅在产前一周、就巢期、产蛋后十天及休产期时PRLR基因在垂体、下丘脑、卵巢三个组织中的表达水平,探讨了三个组织中PRLR基因在不同繁殖时期mRNA的表达规律。 研究发现PRLR基因在下丘脑、垂体、卵巢上均有表达,表达量从高到低依次为:卵巢下丘脑垂体,各组织间PRLR mRNA的表达量存在明显差异,其中卵巢和下丘脑上的表达量差异显著(P0.05),卵巢和垂体上的表达量差异极显著(P0.01)。对不同时期分析结果显示PRLR mRNA表达量从高到低依次是:就巢期产蛋前一周产蛋后十天休产期。其中就巢期PRLR基因的表达量分别是产蛋前一周、产蛋后十天、休产期的4倍、40倍、45倍,差异均极显著(P0.01)。对同一组织不同时期PRLR mRNA表达量分析表明,垂体四个时期间PRLR基因表达量从高到低依次是:就巢期休产期产蛋后十天产蛋前一周,就巢期的PRLR基因的表达量极显著高于其它三个时期(P0.01),其它三个时期之间差异不显著(P0.05);下丘脑四个时期间PRLR基因表达量从高到低依次是:就巢期产蛋后十天产蛋前一周休产期,就巢期和其它三个时期的PRLR基因的表达量差异均极显著(P0.01),其它三个时期之间差异不显著(P0.05);卵巢四个时期PRLR基因的表达量从高到低依次是:就巢期产蛋前一周休产期产蛋后十天,就巢期的PRLR基因的表达量极显著高于其它三个时期(P0.01),产蛋前一周的PRLR基因的表达量高于产蛋后十天和休产期,差异极显著(P0.01)。 在就巢期,三个组织PRLR基因的表达量都很高,之间差异不显著(P0.05)。其中垂体就巢期三个时间段的PRLR基因表达量从高到低依次是:就巢30-31天就巢1-3天就巢14-16天,就巢30-31天PRLR基因的表达量是就巢14-16天的3倍,差异显著(P0.05);下丘脑和卵巢就巢期三个时间段PRLR基因表达量从高到低依次是:就巢1-3天就巢14-16天就巢30-31天,差异不显著(P0.05)。
URL [本文引用: 2]
以浙东白鹅为研究对象,采用克 隆、测序技术获得浙东白鹅PRL基因789 bp的c DNA序列,包括了690 bp的编码区,编码229个氨基酸。并与其他物种进行同源性比较,发现该基因序列与其他鹅PRL序列相比发生了A170G、T315C、C316A、 C318T、T516C、G617A突变;其中A170G发生了氨基酸H→R的变化,C318T处发生了氨基酸H→N的变化,G617A发生了氨基酸 R→H的变化。与禽类PRL基因核苷酸和氨基酸序列同源性均在92.9%以上,与人和哺乳动物同源性在51.8%~77.2%之间。利用实时荧光定量 PCR技术检测鹅产蛋期到就巢期PRL基因在相关组织中的表达变化。在产蛋期和就巢期,垂体中PRL基因表达量显著高于其他3个组织,下丘脑、卵巢、输卵 管3个组织之间PRL基因mRNA表达量没有显著性差异。就巢期组织中PRL基因表达量极显著高于产蛋期同一组织中mRNA表达量。
URL [本文引用: 2]
以浙东白鹅为研究对象,采用克 隆、测序技术获得浙东白鹅PRL基因789 bp的c DNA序列,包括了690 bp的编码区,编码229个氨基酸。并与其他物种进行同源性比较,发现该基因序列与其他鹅PRL序列相比发生了A170G、T315C、C316A、 C318T、T516C、G617A突变;其中A170G发生了氨基酸H→R的变化,C318T处发生了氨基酸H→N的变化,G617A发生了氨基酸 R→H的变化。与禽类PRL基因核苷酸和氨基酸序列同源性均在92.9%以上,与人和哺乳动物同源性在51.8%~77.2%之间。利用实时荧光定量 PCR技术检测鹅产蛋期到就巢期PRL基因在相关组织中的表达变化。在产蛋期和就巢期,垂体中PRL基因表达量显著高于其他3个组织,下丘脑、卵巢、输卵 管3个组织之间PRL基因mRNA表达量没有显著性差异。就巢期组织中PRL基因表达量极显著高于产蛋期同一组织中mRNA表达量。
[D].
[本文引用: 1]
[本文引用: 1]
[D].
[本文引用: 2]
[本文引用: 2]
URL [本文引用: 2]
为探讨狮头鹅不同繁殖阶段FSHβ及其受体的表达规律,采用 SYBR Green Ⅰ荧光定量PCR方法对产蛋期、就巢期和休产期狮头鹅垂体和卵巢组织中FSHβ及其受体的表达水平进行检测.结果表明:FSHβ、FSHR在狮头鹅垂体和 卵巢组织中均有表达,垂体FSHβ的相对表达量极显著高于卵巢(P＜0.01),而FSHR的相对表达量在卵巢中较高；在不同的繁殖阶段,垂体FSHβ与 卵巢FSHR的表达趋势相似,产蛋期相对表达量极显著高于就巢期和休产期(P＜0.01).由此可见,狮头鹅垂体和卵巢组织中FSHβ及FSHR表达水平 的变化与繁殖周期密切相关.
URL [本文引用: 2]
为探讨狮头鹅不同繁殖阶段FSHβ及其受体的表达规律,采用 SYBR Green Ⅰ荧光定量PCR方法对产蛋期、就巢期和休产期狮头鹅垂体和卵巢组织中FSHβ及其受体的表达水平进行检测.结果表明:FSHβ、FSHR在狮头鹅垂体和 卵巢组织中均有表达,垂体FSHβ的相对表达量极显著高于卵巢(P＜0.01),而FSHR的相对表达量在卵巢中较高；在不同的繁殖阶段,垂体FSHβ与 卵巢FSHR的表达趋势相似,产蛋期相对表达量极显著高于就巢期和休产期(P＜0.01).由此可见,狮头鹅垂体和卵巢组织中FSHβ及FSHR表达水平 的变化与繁殖周期密切相关.
URLPMID:26218061 [本文引用: 2]
Gonadotropin-releasing hormone (GnRH), a neuropeptide, plays a vital role in the hypothalamus–pituitary–gonadal (HPG) axis. In vertebrates, GnRH is crucial for the onset of sexual development and the entire reproductive process. The purpose of this study was to identify genetic factors associated with egg-laying traits of Muscovy ducks.The full-length cDNA (474 bp) of Muscovy duckGnRHwas obtained and characterised. It encodes 92 amino acids containing a 1-amino acid signal peptide cleavage site. Phylogenetic analysis revealed that Muscovy duckGnRHhas a close relationship withAnas platyrhynchos GnRH.GnRHshowed significantly different expression profiles between 4 developmental periods in the hypothalamus, pituitary, and ovary. The expression ofGnRHin the laying period (3602weeks) was higher than at other periods in the three tissues.GnRHwas widely expressed in 12 examined tissues of nesting and laying Muscovy ducks. In the hypothalamus, pituitary and gonads, the expression ofGnRHwas higher than in other tissues.In laying Muscovy ducks, the expression ofGnRHin the hypothalamus, pituitary, ovary, muscular stomach, pancreas, heart, duodenum and spleen was significantly higher than in nesting dusks. Differences were detected in the liver and glandular stomach between laying ducks and nesting ducks. Differences between the kidney and lung were not significant.In the pituitary, theGnRHandGnRHreceptor (GnRHR) genes shared the same expression profiles during 4 time points. Both genes had the highest expression at 3602weeks of age.A mutation (g.206G02>02A) in the 500-flanking region was associated with egg-laying performance. Individuals with genotype GG had better egg-laying performance than the individuals with genotype AA.GnRHmay be used as a marker gene for laying performance in the Muscovy duck. Gonadotropin-releasing hormone (GnRH), a neuropeptide, plays a vital role in the hypothalamus–pituitary–gonadal (HPG) axis. In vertebrates, GnRH is crucial for the onset of sexual development and the entire reproductive process. The purpose of this study was to identify genetic factors associated with egg-laying traits of Muscovy ducks. The full-length cDNA (474 bp) of Muscovy duckGnRHwas obtained and characterised. It encodes 92 amino acids containing a 1-amino acid signal peptide cleavage site. Phylogenetic analysis revealed that Muscovy duckGnRHhas a close relationship withAnas platyrhynchos GnRH. GnRHshowed significantly different expression profiles between 4 developmental periods in the hypothalamus, pituitary, and ovary. The expression ofGnRHin the laying period (3602weeks) was higher than at other periods in the three tissues.GnRHwas widely expressed in 12 examined tissues of nesting and laying Muscovy ducks. In the hypothalamus, pituitary and gonads, the expression ofGnRHwas higher than in other tissues. In laying Muscovy ducks, the expression ofGnRHin the hypothalamus, pituitary, ovary, muscular stomach, pancreas, heart, duodenum and spleen was significantly higher than in nesting dusks. Differences were detected in the liver and glandular stomach between laying ducks and nesting ducks. Differences between the kidney and lung were not significant. In the pituitary, theGnRHandGnRHreceptor (GnRHR) genes shared the same expression profiles during 4 time points. Both genes had the highest expression at 3602weeks of age. A mutation (g.206G02>02A) in the 500-flanking region was associated with egg-laying performance. Individuals with genotype GG had better egg-laying performance than the individuals with genotype AA.GnRHmay be used as a marker gene for laying performance in the Muscovy duck.
URLPMID:20199684 [本文引用: 2]
Background The elevation of egg production and the inhibition of incubation behavior are the aims of modern poultry production. Prolactin (PRL) gene is confirmed to be critical for the onset and maintenance of these reproductive behaviors in birds. Through PRL, dopamine D1 receptor (DRD1) was also involved in the regulation of chicken reproductive behavior. However, the genetic effects of this gene on chicken egg production and broodiness have not been studied extensively. The objective of this research was to evaluate the genetic effects of the DRD1 gene on chicken egg production and broodiness traits. Results In this study, the chicken DRD1 gene was screened for the polymorphisms by cloning and sequencing and 29 variations were identified in 3,342 bp length of this gene. Seven single nucleotide polymorphism (SNPs) among these variations, including a non-synonymous mutation (A+505G, Ser169Gly), were located in the coding region and were chosen to analyze their association with chicken egg production and broodiness traits in 644 Ningdu Sanhuang individuals. Two SNPs, G+123A and C+1107T, were significantly associated with chicken broody frequency (P < 0.05). Significant association was also found between the G+1065A - C+1107T haplotypes and chicken broody frequency (P < 0.05). In addition, the haplotypes of G+123A and T+198C were significantly associated with weight of first egg (EW) (P = 0.03). On the other hand, the distribution of the DRD1 mRNA was observed and the expression difference was compared between broodiness and non-broodiness chickens. The DRD1 mRNA was predominantly expressed in subcutaneous fat and abdominal fat of non-broodiness chicken, and then in heart, kidney, oviduct, glandular stomach, hypothalamus, and pituitary. In subcutaneous fat and abdominal fat, the level of non-broodiness was 26 to 28 times higher than that of broodiness. In pituitary, it was 5-fold higher. In heart, oviduct, and kidney, a 2-3 times decrease from non-broodiness to broodiness was displayed. In glandular stomach and hypothalamus, the level seen in non-broodiness and broodiness was almost the same. Conclusion The polymorphisms of the DRD1 gene and their haplotypes were associated with chicken broody frequency and some egg production traits. The mRNA distribution was significant different between broodiness and non-broodiness chickens.
URLPMID:20181857 [本文引用: 2]
Chicken broodiness is a polygenic trait controlled by autosomal genes. Prolactin gene is a candidate of great interest in molecular studies of broodiness. However, another candidate dopamine D2 receptor (DRD2) gene has not been studied extensively. The objective of this study was to analyze the genetic effects of the DRD2 gene on chicken broodiness through linkage disequilibrium analyses, tag SNP selection, genetic diversity observation, 2-tailed test, and association analyses. In this study, we assayed 27 variations of this gene in 456 individuals from 6 chicken populations to observe linkage disequilibrium pattern, the tag SNP, and genetic diversity. Among the 6 populations, Taihe Silkies exhibited no characteristic between the square of the correlation coefficient of gene frequencies (r) and physical distance. The other populations including Red Jungle Fowls, Xinghua chickens, Ningdu Sanhuang chickens (NDH), Baier Huang chickens, and Leghorn layers exhibited conspicuous characteristic of decreasing r value over physical distance. Linkage disequilibrium decayed more rapidly in Red Jungle Fowls, Xinghua, and NDH than in Baier Huang and Leghorn layers. Allelic frequencies and genotype distributions in the 5 populations showed that A-38600G, I-38463D, T-32751C, A-16105G, A-6543G, C-6539T, and A+2794G were possibly associated with broodiness. Besides the above 7 sites, another 2 sites that might be associated with broodiness were screened by 2-tailed test. All 9 sites were used for association analyses with broodiness in 644 NDH chickens. A significant association (P < 0.05) was found between A-16105G and broody frequency (%), and the T+619C in intron 1 was significantly associated with duration of broodiness (P < 0.05). These findings suggested that the DRD2 gene should be included in future genetic studies of chicken broodiness and 2 SNP of A-16105G and T+619C might be markers for breeding against broodiness.
URLPMID:25573700 [本文引用: 2]
61VIP expression and Fos co-expression are extensively associated with nesting behavior.61VIP neurons and nesting are associated throughout the social behavior network.61VIP neurons relate to individual differences in nesting readiness and performance.61VIP and Fos co-expression relates to appetitive and consummatory aspects of behavior.
URLPMID:18420979 [本文引用: 2]
Broodiness is a polygenic trait controlled by a small number of autosomal genes. Vasoactive intestinal peptide receptor-1 (VIPR-1) gene could be a candidate of chicken broodiness, and its genomic variations and genetic effects on chicken broodiness traits were analyzed in this study. The partial cloning and sequencing of the VIPR-1 gene showed that the average nucleotide diversity was 0.00669 +/- 0.00093 in Red Jungle Fowls (RJF), and 0.00582 +/- 0.00026 in domestic chickens. One hundred twenty-eight variation sites were identified in the 11,136-bp region of the chicken VIPR-1 gene. Twenty variation sites were genotyped using PCR-RFLP or PCR method to analyze average diversity, linkage-disequilibrium pattern, and haplotype structure in RJF, Xinghua chickens, Ningdu Sanhuang chickens, Baier Huang chickens, and Leghorn Layers. The RJF, Xinghua, Ningdu Sanhuang, and Baier Huang exhibited distinct characteristic of decreasing r(2) value over physical distance. Haplotype analyses showed that some variation sites of the 27-kb region from exon 6 to exon 11 could be associated with broodiness. The distribution of genotypic and allelic frequencies, and heterozygosities in the above 5 populations showed that A-284G, A+457G, C+598T, D+19820I, C+37454T, C+42913T, and C+53327T might be associated with broodiness. The 7 sites and the other 4 sites were genotyped in 644 NDH individuals under cage condition and were used for association analyses between each site and chicken broodiness traits. A significant association (P < 0.05) was found between C+598T in intron 2 and broody frequency (%). Another significant association (P < 0.05) was found between C+53327T and duration of broodiness, in which allele C was positive for DB.
URLMagsci [本文引用: 2]
利用350至450日龄的就巢粤黄鸡观察多巴胺（DA）和5－羟胺（5－HT）受体拮抗剂氯丙嗪和赛更啶对就巢的影响。就巢就2d开始每天口服氯丙嗪60mg/(kg.d)或赛更啶8mg/(kg.d),7d后或于就巢终止后停止药处理。试验1：13只氯丙嗪处理鸡中92．3％（12／13）于处理后5．4±1．1d终止就巢；15只赛更啶处理鸡有60．0％（9／15）在处理后3．2±0.3d终止就巢；全部（100％）10只同时处理氯丙嗪和赛更啶的鸡于处理后3．2±0.3d终止就巢；8只对照组鸡在不受任何处理时持续就巢，其就巢期被主观为22d。试验2：7只就巢鸡在就巢的2、4、9、14和19d血浆PRL水平维持在200－400mg/ml之间，9只氯丙嗪处理后终止就巢的7只鸡以及13只赛更啶处理后终止就巢的6只鸡，血浆PRL由就巢第2d与对照组类似的民剧下降到第4d小于20mg/ml，并至第19d一直处于较低水平。对照组在持续就巢期间的血浆LH水平低于1．0ng/ml，而用药后上就巢鸡的血浆LH水平从处理前的小于1．0ng/ml逐渐上升至第19天的3．0ng/ml左右。药物处理后仍未终止就巢鸡的血浆PRL和LH水平都与对照组的类似。以上结果说明用DA和5－HT受体拮抗剂氯丙嗪和赛更啶处理后，抑制了垂体PRL的分泌并使 鸡终止就巢，在整体水平证明了下丘脑DA和5－HT两种神经递质对就巢发生具有共的加性促进作用。
URLMagsci [本文引用: 2]
利用350至450日龄的就巢粤黄鸡观察多巴胺（DA）和5－羟胺（5－HT）受体拮抗剂氯丙嗪和赛更啶对就巢的影响。就巢就2d开始每天口服氯丙嗪60mg/(kg.d)或赛更啶8mg/(kg.d),7d后或于就巢终止后停止药处理。试验1：13只氯丙嗪处理鸡中92．3％（12／13）于处理后5．4±1．1d终止就巢；15只赛更啶处理鸡有60．0％（9／15）在处理后3．2±0.3d终止就巢；全部（100％）10只同时处理氯丙嗪和赛更啶的鸡于处理后3．2±0.3d终止就巢；8只对照组鸡在不受任何处理时持续就巢，其就巢期被主观为22d。试验2：7只就巢鸡在就巢的2、4、9、14和19d血浆PRL水平维持在200－400mg/ml之间，9只氯丙嗪处理后终止就巢的7只鸡以及13只赛更啶处理后终止就巢的6只鸡，血浆PRL由就巢第2d与对照组类似的民剧下降到第4d小于20mg/ml，并至第19d一直处于较低水平。对照组在持续就巢期间的血浆LH水平低于1．0ng/ml，而用药后上就巢鸡的血浆LH水平从处理前的小于1．0ng/ml逐渐上升至第19天的3．0ng/ml左右。药物处理后仍未终止就巢鸡的血浆PRL和LH水平都与对照组的类似。以上结果说明用DA和5－HT受体拮抗剂氯丙嗪和赛更啶处理后，抑制了垂体PRL的分泌并使 鸡终止就巢，在整体水平证明了下丘脑DA和5－HT两种神经递质对就巢发生具有共的加性促进作用。
URL [本文引用: 2]
URLPMID:3322132 [本文引用: 2]
Abundant evidence indicates that chicken reproduction is strictly regulated by the hypothalamic-pituitary-gonad (HPG) axis, and the genes included in the HPG axis have been studied extensively. However, the question remains as to whether any other genes outside of the HPG system are involved in regulating chicken reproduction. The present study was aimed to identify, on a genome-wide level, novel genes associated with chicken reproductive traits. Suppressive subtractive hybridization (SSH), genome-wide association study (GWAS), and gene-centric GWAS were used to identify novel genes underlying chicken reproduction. Single marker-trait association analysis with a large population and allelic frequency spectrum analysis were used to confirm the effects of candidate genes. Using two full-sib Ningdu Sanhuang (NDH) chickens,GARNL1was identified as a candidate gene involved in chicken broodiness by SSH analysis. Its expression levels in the hypothalamus and pituitary were significantly higher in brooding chickens than in non-brooding chickens. GWAS analysis with a NDH two tail sample showed that 2802 SNPs were significantly associated with egg number at 300 d of age (EN300). Among the 2802 SNPs, 2 SNPs composed a block overlapping theGARNL1gene. The gene-centric GWAS analysis with another two tail sample of NDH showed thatGARNL1was strongly associated with EN300 and age at first egg (AFE). Single marker-trait association analysis in 1301 female NDH chickens confirmed that variation in this gene was related to EN300 and AFE. The allelic frequency spectrum of the SNP rs15700989 among 5 different populations supported the above associations. Western blotting, RT-PCR, and qPCR were used to analyze alternative splicing of theGARNL1gene. RT-PCR detected 5 transcripts and revealed that the transcript, which has a 141 bp insertion, was expressed in a tissue-specific manner. Our findings demonstrate that theGARNL1gene contributes to chicken reproductive traits.
URLPMID:23405160 [本文引用: 4]
The geese have strong broodiness and poor egg performance. These characteristics are the key issues that hinder the goose industry development. Yet little is known about the mechanisms responsible for follicle development due to lack of genomic resources. Hence, studies based on high-throughput sequencing technologies are needed to produce a comprehensive and integrated genomic resource and to better understand the biological mechanisms of goose follicle development.In this study, we performed de novo transcriptome assembly and gene expression analysis using short-read sequencing technology (Illumina). We obtained 67,315,996 short reads of 100 bp, which were assembled into 130,514 unique sequences by Trinity strategy (mean size鈥=鈥753 bp). Based on BLAST results with known proteins, these analyses identified 52,642 sequences with a cut-off E-value above 10(-5). Assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. In addition, we investigated the transcription changes during the goose laying/broodiness period using a tag-based digital gene expression (DGE) system. We obtained a sequencing depth of over 4.2 million tags per sample and identified a large number of genes associated with follicle development and reproductive biology including cholesterol side-chain cleavage enzyme gene and dopamine beta-hydroxylas gene. We confirm the altered expression levels of the two genes using quantitative real-time PCR (qRT-PCR).The obtained goose transcriptome and DGE profiling data provide comprehensive gene expression information at the transcriptional level that could promote better understanding of the molecular mechanisms underlying follicle development and productivity.
URLPMID:29408859 [本文引用: 10]
Leptin was known as a pivotal regulator for the control of food intake and energy expenditure. However, leptin has also been found to be involved in the regulation of female reproductive system through interactions with pathways in the hypothalamic-hypophyseal axis and direct action at the ovarian level. In the present study, granulosa cells from goose ovarian preovulatory (F1-F3) follicles... [Show full abstract]
URL [本文引用: 3]
试验比较了就巢鸡和产蛋鸡生殖系统发育及催乳素(PRL)和褪黑激素受体1c(Mel-1c)基因表达量的变化。从1296只42周龄商品代北京油鸡中挑选出有典型就巢行为表现的就巢鸡30只和无就巢行为表现的产蛋鸡30只,饲养在两个单独的栏舍内,每舍内15只就巢鸡和15只产蛋鸡,43周龄末对所有试验鸡采血测定血清PRL、促黄体素(LH)和雌激素(E2)含量,每舍内分别选取就巢鸡和产蛋鸡各3只进行屠宰,观察卵巢、输卵管发育情况,并取卵巢和输卵管样品,测定PRL和Mel-1c基因的相对表达量。结果表明:就巢鸡的PRL、LH浓度均显著高于产蛋鸡(P0.05),而E2浓度显著低于产蛋鸡(P0.05);就巢鸡和产蛋鸡在体重、卵巢发育方面有显著差异;就巢鸡卵巢中PRL基因、Mel-1c基因相对表达量显著高于产蛋鸡(P0.05),而输卵管中不同生理时期基因相对表达量差异不显著(P0.05);PRL基因相对表达量在卵巢中显著高于输卵管中(P0.05),Mel-1c基因相对表达量在卵巢和输卵管中差异不显著(P0.05)。
URL [本文引用: 3]
试验比较了就巢鸡和产蛋鸡生殖系统发育及催乳素(PRL)和褪黑激素受体1c(Mel-1c)基因表达量的变化。从1296只42周龄商品代北京油鸡中挑选出有典型就巢行为表现的就巢鸡30只和无就巢行为表现的产蛋鸡30只,饲养在两个单独的栏舍内,每舍内15只就巢鸡和15只产蛋鸡,43周龄末对所有试验鸡采血测定血清PRL、促黄体素(LH)和雌激素(E2)含量,每舍内分别选取就巢鸡和产蛋鸡各3只进行屠宰,观察卵巢、输卵管发育情况,并取卵巢和输卵管样品,测定PRL和Mel-1c基因的相对表达量。结果表明:就巢鸡的PRL、LH浓度均显著高于产蛋鸡(P0.05),而E2浓度显著低于产蛋鸡(P0.05);就巢鸡和产蛋鸡在体重、卵巢发育方面有显著差异;就巢鸡卵巢中PRL基因、Mel-1c基因相对表达量显著高于产蛋鸡(P0.05),而输卵管中不同生理时期基因相对表达量差异不显著(P0.05);PRL基因相对表达量在卵巢中显著高于输卵管中(P0.05),Mel-1c基因相对表达量在卵巢和输卵管中差异不显著(P0.05)。
URLMagsci [本文引用: 2]
<FONT face=Verdana>旨在利用抑制消减杂交技术构建鹅就巢与产蛋cDNA基因文库，筛选就巢母鹅上调与下调表达的基因。以就巢期和产蛋期鹅卵巢组织互为检测子和驱动子, 进行正反双向消减杂交, 获得鹅卵巢差异表达基因文库。从双向文库随机挑选单菌落进行PCR验证，结果表明文库质量良好。选取654个阳性克隆进行测序分析,获得641条表达序列标签(ESTs)。对ESTs进行去除载体序列、质量检测、聚类及拼接，经BLASTn比对，有166条ESTs找到与之匹配的同源序列，其中92个是功能基因。比较就巢与产蛋SSH文库，发现就巢SSH库中参与细胞凋亡、信号转导及细胞结构的基因出现频率较高；产蛋特异性基因文库中参与物质能量代谢、细胞防御的基因较多；尚有许多差异片段属于未知功能蛋白或理论推定蛋白。结果提示，催乳素受体、抗苗勒管激素以及雌激素受体基因在就巢期上调表达可能与鹅就巢行为的启动与维持有关。该结果为进一步研究鹅就巢行为分子调控机制奠定基础。</FONT>
URLMagsci [本文引用: 2]
<FONT face=Verdana>旨在利用抑制消减杂交技术构建鹅就巢与产蛋cDNA基因文库，筛选就巢母鹅上调与下调表达的基因。以就巢期和产蛋期鹅卵巢组织互为检测子和驱动子, 进行正反双向消减杂交, 获得鹅卵巢差异表达基因文库。从双向文库随机挑选单菌落进行PCR验证，结果表明文库质量良好。选取654个阳性克隆进行测序分析,获得641条表达序列标签(ESTs)。对ESTs进行去除载体序列、质量检测、聚类及拼接，经BLASTn比对，有166条ESTs找到与之匹配的同源序列，其中92个是功能基因。比较就巢与产蛋SSH文库，发现就巢SSH库中参与细胞凋亡、信号转导及细胞结构的基因出现频率较高；产蛋特异性基因文库中参与物质能量代谢、细胞防御的基因较多；尚有许多差异片段属于未知功能蛋白或理论推定蛋白。结果提示，催乳素受体、抗苗勒管激素以及雌激素受体基因在就巢期上调表达可能与鹅就巢行为的启动与维持有关。该结果为进一步研究鹅就巢行为分子调控机制奠定基础。</FONT>
URLPMID:28866373 [本文引用: 2]
Broodiness causes reduced reproductive ability in poultry, but its regulatory mechanism remains poorly understood. ROS (reactive oxygen species) and autophagy are important for follicular development, and the interaction between the two may play a role in regulating broodiness. We examined goose follicles for ROS and oxidation scavenger activities during the egg-laying and broody stages. The follicular granulosa cells were exposed to media containing H 2 O 2 , and the interactions between ROS and autophagy in follicular granulosa cells in vitro were analyzed using a Western blot method. We found that the activities of superoxide dismutase ( SOD ) and lactate dehydrogenase ( LDH ) were enhanced and the amount of malondialdehyde ( MDA ) decreased in broody goose follicles. H 2 O 2 inhibited the cell viability and induced autophagy. Furthermore, it was also found that H 2 O 2 regulated autophagy by reducing mTOR and increasing p53 ; however, H 2 O 2 had no impact on Beclin1 or ATG12 . It was also shown that the enhanced autophagy lessened ROS-induced damages. We conclude that ROS and autophagy both played important roles in regulating follicular development to control broodiness in geese, and ROS activated autophagy in follicular granulosa cells via the mTOR pathway.
URLPMID:3989219 [本文引用: 4]
Identifying signatures of selection can provide valuable insight about the genes or genomic regions that are or have beenunder selective pressure, which can lead to a better understanding of genotype-phenotype relationships. A commonstrategy for selection signature detection is to compare samples from several populations and search for genomic regionswith outstanding genetic differentiation. Wright鈥檚 fixation index, FST, is a useful index for evaluation of geneticdifferentiation between populations. The aim of this study was to detect selective signatures between different chickengroups based on SNP-wise FST calculation. A total of 96 individuals of three commercial layer breeds and 14 non-commercialfancy breeds were genotyped with three different 600K SNP-chips. After filtering a total of 1 million SNPs were available forFST calculation. Averages of FST values were calculated for overlapping windows. Comparisons of these were thenconducted between commercial egg layers and non-commercial fancy breeds, as well as between white egg layers andbrown egg layers. Comparing non-commercial and commercial breeds resulted in the detection of 630 selective signatures,while 656 selective signatures were detected in the comparison between the commercial egg-layer breeds. Annotation ofselection signature regions revealed various genes corresponding to productions traits, for which layer breeds wereselected. Among them were NCOA1, SREBF2 and RALGAPA1 associated with reproductive traits, broodiness and eggproduction. Furthermore, several of the detected genes were associated with growth and carcass traits, including POMC,PRKAB2, SPP1, IGF2, CAPN1, TGFb2 and IGFBP2. Our approach demonstrates that including different populations with aspecific breeding history can provide a unique opportunity for a better understanding of farm animal selection.
URLMagsci [本文引用: 2]
催乳素（Prolactin，PRL）、生长激素（Growth Hormone，GH）、促甲状腺素β（Thyroid-stimulating Hormone-β，TSHβ）是家禽垂体前叶分泌的具有重要生理功能的激素，这些激素通过调控家禽就巢性的发生与维持、生长发育、免疫应答等过程来影响家禽的产蛋性能、生长性能以及免疫力。垂体特异转录因子POU1F1（Pituitary-specific Transcription Factor）是家禽垂体前叶特异表达的一种具有重要功能的转录因子，它与PRL基因、GH基因、TSHβ基因以及Pit-1自身的启动子结合，调控这些基因的转录，通过调节这些基因的表达而对家禽的生长发育、繁殖等很多方面起着重要的作用。本文比较了家禽Pit-1基因的结构与哺乳动物的差异，阐述了家禽Pit-1基因的表达特点及其生物学效应。考虑到POU1F1的重要作用和作者的研究，作者认为Pit-1基因可作为家禽生长、产蛋、就巢性等重要经济性状的候选基因。
URLMagsci [本文引用: 2]
催乳素（Prolactin，PRL）、生长激素（Growth Hormone，GH）、促甲状腺素β（Thyroid-stimulating Hormone-β，TSHβ）是家禽垂体前叶分泌的具有重要生理功能的激素，这些激素通过调控家禽就巢性的发生与维持、生长发育、免疫应答等过程来影响家禽的产蛋性能、生长性能以及免疫力。垂体特异转录因子POU1F1（Pituitary-specific Transcription Factor）是家禽垂体前叶特异表达的一种具有重要功能的转录因子，它与PRL基因、GH基因、TSHβ基因以及Pit-1自身的启动子结合，调控这些基因的转录，通过调节这些基因的表达而对家禽的生长发育、繁殖等很多方面起着重要的作用。本文比较了家禽Pit-1基因的结构与哺乳动物的差异，阐述了家禽Pit-1基因的表达特点及其生物学效应。考虑到POU1F1的重要作用和作者的研究，作者认为Pit-1基因可作为家禽生长、产蛋、就巢性等重要经济性状的候选基因。
URLPMID:3913702 [本文引用: 9]
Recent functional studies have demonstrated that the microRNAs (miRNAs) play critical roles in ovarian gonadal development, steroidogenesis, apoptosis, and ovulation in mammals. However, little is known about the involvement of miRNAs in the ovarian function of fowl. The goose (Anas cygnoides) is a commercially important food that is cultivated widely in China but the goose industry has been hampered by high broodiness and poor egg laying performance, which are influenced by ovarian function.In this study, the miRNA transcriptomes of ovaries from laying and broody geese were profiled using Solexa deep sequencing and bioinformatics was used to determine differential expression of the miRNAs. As a result, 11,350,396 and 9,890,887 clean reads were obtained in laying and broodiness goose, respectively, and 1,328 conserved known miRNAs and 22 novel potential miRNA candidates were identified. A total of 353 conserved microRNAs were significantly differentially expressed between laying and broody ovaries. Compared with miRNA expression in the laying ovary, 127 miRNAs were up-regulated and 126 miRNAs were down-regulated in the ovary of broody birds. A subset of the differentially expressed miRNAs (G-miR-320, G-miR-202, G-miR-146, and G-miR-143*) were validated using real-time quantitative PCR. In addition, 130,458 annotated mRNA transcripts were identified as putative target genes. Gene ontology annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that the differentially expressed miRNAs are involved in ovarian function, including hormone secretion, reproduction processes and so on.The present study provides the first global miRNA transcriptome data in A. cygnoides and identifies novel and known miRNAs that are differentially expressed between the ovaries of laying and broody geese. These findings contribute to our understanding of the functional involvement of miRNAs in the broody period of goose.
URLPMID:24469721 [本文引用: 8]
MicroRNAs (miRNAs) are endogenous small noncoding RNAs plays a critical role in posttranscriptional regulation of gene expression. Broodiness is observed in most avian species and influences egg production. Several genes are known to play an important role in regulating the progress of reproduction. The goose is one of the most important waterfowls. However, the involvement of miRNAs in the broodiness behavior of Anser cygnoides (Swan Goose) is unknown. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in egg-laying and brooding behavior of geese in our study. The results showed 38 up-regulated and 14 down-regulated known miRNAs/miRNA*s with reads >1,000 in at least one group and a fold change of >2.0, compared with those of the egg-laying group ( P <0.001). We also identified 114 and 94 novel miRNAs in the broody and egg-laying groups, respectively. Of these, 4 novel miRNAs were differentially expressed between the two groups. The study showed the expression of small RNAs in goose reproduction and identified known and novel miRNAs regulated in broodiness. The results reveal that these differentially expressed miRNAs may be involved in broodiness of A. cygnoides .
URLPMID:27199452 [本文引用: 7]
Broodiness is the primary factor influencing egg production in geese, in which several genes and miRNAs participate. Detailed spatiotemporal profiles of miRNAs encompassing follicle development levels, however, are lacking. In this study, we collected preovulatory follicles (classified as small white follicles, large white follicles, and small yellow follicles) from brooding and laying geese and aimed to analyze microRNA (miRNA or miR) during folliculogenesis. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in follicle development. The let7 family, miR-10 family, and miR-143 family were abundant in these libraries, and they have been suggested to play a housekeeping role during folliculogenesis. Joint comparisons revealed 23 upregulated and 21 downregulated miRNAs (in at least two comparisons of follicles during brooding and laying,P< 0.1) in the laying stage. Unlike reproduction pathways reported for ovaries, GO and KEGG analysis suggested pathways for cell apoptosis and proliferation, such as the regulation of actin cytoskeleton, endocytosis, axon guidance, pathways in cancer, tight junctions, focal adhesion, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and the Wnt signaling pathway in folliculogenesis. This study revealed the miRNAs that were directly involved in follicular atresia, and our results added to the understanding of the functional involvement of miRNAs during specific stages of follicle development.
URLPMID:23666971 [本文引用: 3]
Context: Human follicular fluid is a combination of proteins, metabolites, and ionic compounds that is indicative of the general state of follicular metabolism and is associated with maturation and quality of oocytes. Deviations in these components are often associated with reproductive diseases. There has been no report of microRNAs (miRNAs) in human follicular fluids.Objective: We hypothesized that human follicular fluid may contain miRNAs. We sought to identify cell-free miRNAs in human follicular fluid and to investigate the function of these miRNAs in vitro and any roles they play in polycystic ovary syndrome (PCOS).Design: Genome-wide deep sequencing and TaqMan miRNA arrays were used to identify miRNAs, and the roles of the highly expressed miRNAs in steroidogenesis were investigated in KGN cells. Quantification of candidate miRNAs in follicular fluids of PCOS and controls was performed using TaqMan miRNA assays.Results: We identified miRNAs in microvesicles and the supernatant of human follicular fluid. Bioinformatics analysis showed that the most highly expressed miRNAs targeted genes associated with reproductive, endocrine, and metabolic processes. We found that miR-132, miR-320, miR-520c-3p, miR-24, and miR-222 regulate estradiol concentrations and that miR-24, miR-193b, and miR-483-5p regulate progesterone concentrations. Finally, we showed that miR-132 and miR-320 are expressed at significantly lower levels in the follicular fluid of polycystic ovary patients than in healthy controls (P = .005 and P = .0098, respectively).Conclusion: These results demonstrate that there are numerous miRNAs in human follicular fluids, some of which play important roles in steroidogenesis and PCOS. This study substantially revises our understanding of the content of human follicular fluid and lays the foundation for the future investigation of the role of miRNAs in PCOS.
URLPMID:4539686 [本文引用: 2]
Background Estrogen synthesis is an important function of the mammalian ovary. Estrogen plays important roles in many biological processes, including follicular development, oocyte maturation and endometrial proliferation, and dysfunctions in estrogen synthesis contribute to the development of polycystic ovary syndrome and premature ovarian failure. Classical signaling cascades triggered by follicle-stimulating hormone induce estrogen synthesis via the upregulation of Cyp19a1 in granulosa cells (GCs). This study aimed to determine the effect of microRNA-132 (miR-132) on estradiol synthesis in GCs. Methods Primary mouse GCs were collected from ovaries of 21-day-old immature ICR mice through follicle puncture. GCs were cultured and treated with the stable cyclic adenosine monophosphate analog 8-Br-cAMP or transfected with miR-132 mimics, Nurr1-specific small interfering RNA oligonucleotides and Flag-Nurr1 plasmids. Concentrations of estradiol and progesterone in culture medium were determined by an automated chemiluminescence-based assay. Quantitative real time PCR and western blot were performed to identify the effect of miR-132 on Cyp19a1, Cyp11a1 and an orphan nuclear receptor-Nurr1 expression in GCs. Direct suppression of Nurr1 via its 3'-untranslated region by miR-132 were further verified using luciferase reporter assays. Results The expression level of miR-132 in cultured mouse GCs was significantly elevated during 48 h of treatment with 8-Br-cAMP. The synthesis of estradiol increased after the overexpression of miR-132 in mouse GCs. The real-time PCR results demonstrated that miR-132 induced the expression of Cyp19a1 significantly. Nurr1, an orphan nuclear receptor that suppresses Cyp19a1 expression, was found to be a direct target of miR-132. Nurr1 was suppressed by miR-132, as indicated by a luciferase assay and Western blotting. The knockdown of Nurr1 primarily elevated the synthesis of estradiol and partially attenuated the miR-132-induced estradiol elevation, and the ectopic expression of Flag-Nurr1 abrogated the stimulatory effect of miR-132 on estradiol synthesis in mouse GCs. Conclusions Our findings suggest that miR-132 is involved in the cAMP signaling pathway and promotes estradiol synthesis via the translational repression of Nurr1 in ovarian GCs.
URLPMID:27965096 [本文引用: 2]
Deregulation of epigenetic modification by microRNAs (miRNAs) contributes to the development of estrogen deficiency, a hallmark of the multigenic endocrine disorder polycystic ovary syndrome (PCOS), but its etiology remains unclear. Previous study has pointed to a tight association between miR-320a expression and oocyte development in human follicular fluid. Given that the bi-directional communication existing between cumulus cells (CCs) and follicular fluid is essential for ovarian steroidogenesis and CCs are the main site where estrogen is finally synthesized, it is intriguing to know whether miR-320a have any regulatory roles in this unique cell. Here we report that miR-320a expression is significantly down-regulated in primary CCs from PCOS patients and this down-regulation promotes estrogen deficiency in CCs. From a mechanistic standpoint, IGF1 regulates miR-320a expression in CCs, and miR-320a could potentiate the steroidogenesis in CCs through modulation of CYP11A1 and CYP19A1 expression, by directly targeting 3'untranslated region (3'UTR) of the osteogenic transcription factor RUNX2. Overall, our results strongly suggest that deregulation of miR-320a/RUNX2/CYP11A1 (CYP19A1) cascade plays an important role in the development of estrogen deficiency in human CCs. Testing patients for miR-320a/RUNX2 expression ratios may provide more accurate diagnostic information and could influence the recommended course of treatment for PCOS.
URLPMID:21846797 [本文引用: 2]
Abstract Estradiol is a steroid hormone that not only plays an important role in ovarian follicular development but also is associated with many reproductive disorders. Owing to the importance of aromatase in the production of estradiol, the regulation of aromatase gene expression at the transcriptional level has been an extensive area of study for over two decades. However, its regulation at the posttranscriptional level has remained unclear. Here, we show that micro-RNA378 (miR-378) is spatiotemporally expressed in porcine granulosa cells, the cells that generate estradiol in the ovary during follicular development, in an inverse manner compared with the expression of aromatase. In vitro overexpression and inhibition experiments revealed that aromatase expression, and therefore estradiol production, by granulosa cells, is posttranscriptionally down-regulated by miR-378. Furthermore, site-directed mutation studies identified two binding sites in the 3'-untranslated region (3'-UTR) of the aromatase coding sequence that are critical for the action of miR-378. Interestingly, overexpression of the aromatase 3'-UTR enhanced aromatase expression at the protein level in granulosa cells, possibly mediated by the binding of miR-378 within this region, thereby reducing the binding of this micro-RNA to the endogenous aromatase 3'-UTR.
URLPMID:20118412 [本文引用: 3]
Many members of the TGF-β superfamily are indicated to play important roles in ovarian follicular development, such as affecting granulosa cell function and oocyte maturation. Abnormalities associated with TGF-β1 signaling transduction could result in female infertility. MicroRNAs (miRNAs), as small noncoding RNAs, were recently found to regulate gene expression at posttranscriptional levels. However, little is known about the role of miRNAs in TGF-β-mediated granulosa cell proliferation and granulosa cell function. In this study, the miRNA expression profiling was identified from TGF-β1-treated mouse preantral granulosa cells (GCs), and three miRNAs were found to be significantly up-regulated and 13 miRNAs were down-regulated. Among up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGFβ superfamily type I receptors, thus blocking phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by TGF-β1/Smads pathway. The ectopic expression of miR-224 can enhance TGF-β1-induced GC proliferation through targeting Smad4. Inhibition of endogenous miR-224 partially suppressed GC proliferation induced by TGF-β1. In addition, both miR-224 and TGF-β1 can promote estradiol release from GC, at least in part, through increasing CYP19A1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF-β1-induced GC proliferation and ovarian estrogen release. Such miRNA-mediated effects could be potentially used for regulation of reproductive processes or for treatment of reproductive disorders.
URLPMID:28765643 [本文引用: 1]
Abstract Gametogenesis is a complicated biological process by which sperm and egg are produced for genetic transmission between generations. In many animals, the germline is segregated from the somatic lineage in early embryonic development through the specification of primordial germ cells (PGCs), the precursors of gametes for reproduction and fertility. In some species, such as fruit fly and zebrafish, PGCs are determined by the maternally provided germ plasm which contains various RNAs and proteins. Here, we identified a germ plasm/PGC-specific microRNA miR-202-5p for the first time in zebrafish. MiR-202-5p was specifically expressed in gonad. In female, it was expressed and accumulated in oocytes during oogenesis. Quantitative reverse transcription PCR and whole mount in situ hybridization results indicated that miR-202-5p exhibited a typical germ plasm /PGC-specific expression pattern throughout embryogenesis, which was consistent with that of the PGC marker vasa, indicating that miR-202-5p was a component of germ plasm and a potential PGC marker in zebrafish. Our present study might be served as a foundation for further investigating the regulative roles of miRNAs in germ plasm formation and PGC development in zebrafish and other teleost.
URL [本文引用: 2]
Female gamete production relies on coordinated molecular and cellular processes that occur in the ovary throughout oogenesis. In fish, as in other vertebrates, these processes have been extensively studied both in terms of endocrine/paracrine regulation and protein expression and activity. The role of small non-coding RNAs in the regulation of animal reproduction remains however largely unknown and poorly investigated, despite a growing interest for the importance of miRNAs in a wide variety of biological processes. Here, we analyzed the role of miR-202, a miRNA predominantly expressed in male and female gonads in several vertebrate species. We studied its expression in the medaka ovary and generated a mutant line (using CRISPR/Cas9 genome engineering) to determine its importance for reproductive success with special interest for egg production. Our results show that miR-202-5p is the biologically active form of the miRNA and that it is expressed in granulosa cells and in the unfertilized egg. The knock out (KO) of miR-202 resulted in a strong phenotype both in terms of number and quality of eggs produced. Mutant females exhibited either no egg production or produced a drastically reduced number of eggs that could not be fertilized, ultimately leading to no reproductive success. We quantified the size distribution of the oocytes in the ovary of KO females and performed a genome-wide transcriptomic analysis approach to identified dysregulated molecular pathways. Together, cellular and molecular analyses indicate that lack of miR-202 impairs the early steps of oogenesis/folliculogenesis and decreases the number of large (i.e. vitellogenic) follicles, ultimately leading to dramatically reduced female fecundity. This study sheds new light on the regulatory mechanisms that control the early steps of follicular development and provides the first in vivo functional evidence that an ovarian-predominant microRNA may have a major role in female reproduction.
URLPMID:21389341 [本文引用: 1]
Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.
[本文引用: 2]
URLPMID:4140302 [本文引用: 2]
Abstract Our previous studies have shown that microRNA-320 (miR-320) is one of the most down-regulated microRNAs (miRNA) in mouse ovarian granulosa cells (GCs) after TGF-脦虏1 treatment. However, the underlying mechanisms of miR-320 involved in GC function during follicular development remain unknown. In this study, we found that pregnant mare serum gonadotropin treatment resulted in the suppression of miR-320 expression in a time-dependent manner. miR-320 was mainly expressed in GCs and oocytes of mouse ovarian follicles in follicular development. Overexpression of miR-320 inhibited estradiol synthesis and proliferation of GCs through targeting E2F1 and SF-1. E2F1/SF-1 mediated miR-320-induced suppression of GC proliferation and of GC steroidogenesis. FSH down-regulated the expression of miR-320 and regulated the function of miR-320 in mouse GCs. miR-383 promoted the expression of miR-320 and enhanced miR-320-mediated suppression of GC proliferation. Injection of miR-320 into the ovaries of mice partially promoted the production of testosterone and progesterone but inhibited estradiol release in vivo. Moreover, the expression of miR-320 and miR-383 was up-regulated in the follicular fluid of polycystic ovarian syndrome patients, although the expression of E2F1 and SF-1 was down-regulated in GCs. These data demonstrated that miR-320 regulates the proliferation and steroid production by targeting E2F1 and SF-1 in the follicular development. Understanding the regulation of miRNA biogenesis and function in the follicular development will potentiate the usefulness of miRNA in the treatment of reproduction and some steroid-related disorders. 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
URLPMID:23813447 [本文引用: 2]
Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle anovulatory wave (n=6 mares). Follicular fluid levels of progesterone and estradiol were lower (P<0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P<0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P <= 0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24 h after (L) administration of an ovulatory dose of hCG (n=5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (P=0.05) and lower granulosa cell levels of CYP19A1 and LHCGR (P<0.005). Levels of miR-21, miR-132, miR-212, and miR-224 were increased (P<0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets, PTEN, RASA1, and SMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132, and miR-212 in the regulation of cell survival, steroidogenesis, and differentiation during follicle selection and ovulation in the monovular ovary.
URLPMID:28876086 [本文引用: 1]
Abstract Ovarian theca cells play an indispensable role in ovarian follicular development and hormone secretion. miR-26a-5p was reported to be differentially expressed in mature and immature chicken ovaries in our previous study; however, the role of miR-26a-5p in regulating ovarian follicle function is still unclear. In this study, we demonstrated that the expression dynamics of TNRC6A mRNA in either chicken ovaries or follicles showed an opposite trend compared with that of chicken miR-26a-5p expression. miR-26a-5p inhibited TNRC6A mRNA expression by directly targeting its 3'-untranslated region in cultured chicken theca cells. Overexpression of miR-26a-5p promoted chicken follicular theca cell proliferation in vitro. Furthermore, overexpression of miR-26a-5p and knockdown of TNRC6A significantly upregulated the antiapoptotic BCL-2 gene. Taken together, this study revealed the expression dynamics of miR-26a-5p and TNRC6A in chicken ovaries and ovarian follicles and the relationship between the expression of miR-26a-5p and TNRC6A in chicken ovarian theca cells. These results suggest that miR-26a-5p facilitates chicken ovarian theca cell proliferation by targeting the TNRC6A gene.
URL [本文引用: 1]
MicroRNAs(miRNAs), small non-coding RNAs, are involved in many aspects of biological processes. Previous studies have indicated that mi RNAs are important for hair follicle development and growth. In our study, we found by q RT-PCR that miR-148 b was signi ficantly upregulated in sheep wool follicle bulbs in anagen phase compared with the telogen phase of the hair follicle cycle. Overexpression of miR-148 b promoted proliferation of both HHDPC and HHGMC. By using the TOPFlash system we demonstrated that miR-148 b could activate Wnt/尾-catenin pathway and b-catenin, cyc D, c-jun and PPARD were consistently upregulated accordingly.Furthermore, transcript factor nuclear factor of activated T cells type 5(NFAT5) and Wnt10 b were predicted to be the target of mi R-148 b and this was substantiated using a Dual-Luciferase reporter system. Subsequently NFAT5 was further identi fied as the target of mi R-148 b using western blotting. These results were considered to indicate that mi R-148 b could activate the Wnt/尾-catenin signal pathway by targeting NFAT5 to promote the proliferation of human hair follicle cells.
URL [本文引用: 1]
miR-214 plays a major role in the self-renewal of skin tissue. However, whether miR-214 regulates the proliferation and differentiation of human hair follicle stem cells (HFSCs) is unknown. Primary...
URLPMID:28473352 [本文引用: 2]
Substantive evidence has indicated that the sympathetic innervation contributes to the regulation and development of ovarian functions. Norepinephrine (NE) is one of the major neurotransmitters contained in the extrinsic ovarian sympathetic nerves and is thought to be a potent moderator of ovarian functions such as steroidogenesis and granulosa cell proliferation or apoptosis. However, the mechanisms of NE regulation of granulosa cell apoptosis in the rat ovary are rare. Real-time PCR and Western blot results show that NE regulates the expression of miR-21 in primary granulosa cells in a dose-dependent manner. Additionally, we found that miR-21 is involved in NE-mediated rat granulosa cells apoptosis and blocks granulosa cell apoptosis by targeting Smad7, a transforming growth factor-beta-inducible mediator of apoptosis in granulosa cells. In primary granulosa cells, a combined treatment of NE and TGF-尾 increased apoptosis and decreased miR-21 expression, but increased SMAD7 protein levels. We also demonstrated that NE regulates miR-21 by coupling to 伪1A-adrenergic receptor (伪1A-AR). This is the first demonstration that NE controls the reproductive functions by modulating the expression of miR-21 and promoting TGF-尾-induced granulosa cell apoptosis. Such NE-mediated effects could be potentially used for regulating the reproductive processes and for treating reproductive disorders.
URLPMID:25342468 [本文引用: 2]
Abstract RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2β (Tra2β) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2β antibody and microarray analysis identified a subset of Tra2β-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2β knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2β knockdown accelerated the decay of BCL2α mRNA that encodes Bcl-2 and full-length 3' UTR, while it did not affect the stability of BCL2β mRNA having a short, alternatively spliced 3' UTR different from BCL2α 3' UTR. RIP assays with anti-Tra2β and anti-Argonaute 2 antibodies, respectively, showed that Tra2β bound to BCL2α 3' UTR, and that Tra2β knockdown facilitated association of miR-204 with BCL2α 3' UTR. The consensus sequence (GAA) for Tra2β-binding lies within the miR-204-binding site of BCL2 3' UTR. Mutation of the consensus sequence canceled the binding of Tra2β to BCL2 3' UTR without disrupting miR-204-binding to BCL2 3' UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2β-silenced or overexpressed cells revealed that Tra2β antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2β mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2β knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2β regulates apoptosis by modulating Bcl-2 expression through its competition with miR-204. This novel function may have a crucial role in tumor growth.Cell Death and Differentiation advance online publication, 24 October 2014; doi:10.1038/cdd.2014.176.
[本文引用: 3]
URLPMID:24297308 [本文引用: 2]
MicroRNAs were reported to be involved in the modulation of tumor development. The aim of our study was to investigate the effect of miR-205 on proliferation and apoptosis of laryngeal squamous cell carcinoma (LSCC) and seek associations between miR-205 and Bcl-2 using in vitro and in vivo methods. Real-time qPCR was used to analyze the expression of miR-205 in LSCC samples and Hep-2 cell line. Apoptosis, cell cycle, and proliferation (MTT) assays were performed to test the apoptosis and proliferation of LSCC cells after miR-205 transfection. Bcl-2 expression in cells was assessed with Western blotting. The tumorigenicity of LSCC cells was evaluated in nude mice model. MiR-205 was significantly down-regulated in LSCC tissues compared to adjacent normal tissues. Lower expression of miR-205 was indicated to be statistically related with advanced clinical stage and T3鈥4 grades. We found that restoration of miR-205 down-regulated the proliferative markers of dihydrofolate reductase and proliferating cell nuclear antigen and apoptotic regulator of Bcl-2. The findings in vitro and in vivo showed miR-205 could suppress cell proliferation and induce cell apoptosis. In addition, Bcl-2 was identified as one of the direct targets of miR-205 in LSCC cells. These results suggest that miR-205 may play as a tumor suppressor in LSCC, probably by targeting Bcl-2 and serve as a potential target for therapeutic intervention.
URLPMID:4686573 [本文引用: 1]
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression. They have important roles in kidney development, homeostasis and disease, and participate in the onset and progression of tubulointerstitial sclerosis and end-stage glomerular lesions that occur in various forms of chronic kidney disease (CKD). In the present study, we elucidated the role of microRNA 205 (miR-205) in cisplatin-induced renal cell apoptosis and explored the molecular mechanisms. The chronic interstitial nephropathy rat model was induced, and the miRNA expression profile in the kidney cells from rats with CKD was screened. Cisplatin-induced apoptosis in normal renal HK-2 cells was evaluated using flow cytometry, and regulation of miR-205 on target gene was validated using luciferase assay, western blot and real time PCR assays. We found that miR-205 expression was significantly decreased in the cells from kidney of CKD rat (P<0.01). Our data showed that when miR-205 was overexpressed or silenced using the mimic or inhibitor, the percentages of apoptotic cells were suppressed or increased significantly (P<0.05), respectively. Moreover, we have identified CMTM4 gene, which is involved in cell proliferation and apoptosis, as a novel target for miR-205. In addition, miR-205 could inhibit apoptosis by binding to the 3鈥橴TR of CMTM4 mRNA and inhibiting its transcriptional activity. This study elucidated that miR-205 plays an important role in the regulation of apoptosis in renal cells, suggesting a potential therapeutic target to hinder CKD development.
[D].
[本文引用: 2]
[本文引用: 2]
PMID:28112253 [本文引用: 2]
Granulosa cells (GCs) are essential somatic cells in the ovary and play an important role in folliculogenesis. Brain-derived neurotropic factor (BDNF) and the TGF-β pathway have been identified as a critical hormone and signalling pathway, respectively, in GCs. In this study, we found that a conserved microRNA family that includes miR-10a and miR-10b repressed proliferation and induced apoptosis in human, mouse, and rat GCs (hGCs, mGCs and rGCs, respectively). Moreover, essential hormones and growth factors in the follicle, such as FSH, FGF9 and some ligands in the TGF-β pathway (TGFβ1, Activin A, BMP4 and BMP15), inhibited miR-10a and miR-10b expression in GCs. In contrast, the miR-10 family suppressed many key genes in the TGF-β pathway, suggesting a negative feedback loop between the miR-10 family and the TGF-β pathway in GCs. By using bioinformatics approaches, RNA-seq, qPCR, FISH, immunofluorescence, Western blot and luciferase reporter assays, BDNF was identified as a direct target of the miR-10 family in GCs. Additionally, reintroduction of BDNF rescued the effects of miR-10a and miR-10b in GCs. Collectively, miR-10a and miR-10b repressed GC development during folliculogenesis by repressing BDNF and the TGF-β pathway. These effects by the miR-10 family on GCs are conserved among different species.
URLPMID:29274780 [本文引用: 1]
Follicle-stimulating hormone (FSH) levels increase estrogen biosynthesis in obese menopausal women. Ovariectomized mice and 3T3-L1 cells were used to explore estrogen biosynthesis in the decline of ovarian function. After ovariectomy, lipid deposition, and FSH and estrogen levels changed, and feed intake increased significantly. In mouse adipose tissue, FSH was found to have a role in accelerating lipid deposition via the peroxisome proliferators-activated receptor pathway, and in inducing estrogen biosynthesis via the steroid hormone metabolism pathway. Furthermore, FSH bound to the FSH receptor promoted CREB phosphorylation, which was activated by cAMP-PKA. Moreover, pCREB could up-regulate PPAR纬 and SREBP2 mRNA levels, resulting in an increased transformation of cholesterol to estrogen. Overall, this study shows that FSH induces fat deposition and promotes the transformation of cholesterol to estrogen through CREB activation by cAMP-PKA in mouse adipose tissue. Our findings provide a new understanding of menopause treatment.
URLPMID:16002539 [本文引用: 1]
Abstract In the present study, we started out to test whether the follicle-stimulating hormone (FSH)-activated p38 MAPK signaling cascade was involved in the regulation of steroidogenesis in granulosa cells (GCs). GCs were prepared from the ovaries of DES-treated immature rats and cultured in serum-free medium. Treatment of GCs with FSH (50 ng/ml) induced the phosphorylation of p38 MAPK rapidly with the phosphorylation being observed within 5 min and reaching the highest level at 30 min. Such activation was protein kinase A-dependent as indicated by the results using specific inhibitors. FSH stimulated the production of progesterone and estradiol as well as the expression of the steroidogenic acute regulatory protein (StAR) in a time-dependent manner, with a maximum level being observed in the production of progesterone and StAR at 48 h. Moreover, the potent p38 MAPK inhibitor SB203580 (20 microM) augmented FSH-induced progesterone and StAR production, while reduced FSH-induced estradiol production at the same time (P<0.01). RT-PCR data showed that inclusion of SB203580 in the media enhanced FSH-stimulated StAR mRNA production, while decreased the FSH-stimulated P450arom mRNA expression (P<0.05). Immunocytochemical studies showed that FSH treatment together with the inhibition of p38 MAPK activity resulted in a higher expression of StAR in mitochondria than FSH treatment alone. FSH also significantly up-regulated the protein level of LRH-1, a member of the orphan receptor family that activates the expression of P450arom in ovaries and testes. p38 MAPK inactivation down-regulated the basal and FSH-induced LRH-1 expression significantly. The intra-cellular level of DAX-1, another orphan receptor that inhibits StAR expression, also decreased upon p38 MAPK being inactivated. For the first time, the present study suggests that FSH-activated p38 MAPK signal pathway regulates progesterone and estrogen production in GCs differentially, and that the transcription factors LRH-1 and DAX-1 might play important roles in the process.
URLPMID:21586554 [本文引用: 2]
Abstract The TGF-β superfamily members are indicated to play key roles in ovarian follicular development, such as granulosa cell proliferation, estrogens, and progesterone production. However, little is known about the roles of TGF-β3 in follicular development. In this study, we found that TGF-β3 was predominantly expressed in granulosa cells of mouse ovarian follicles, and it significantly promoted 17β-estradiol (E(2)) release in a dose-dependent manner. The orphan nuclear receptor steroidogenic factor-1 (SF-1) was required in TGF-β3-induced Cyp19a1 (a key rate-limiting enzyme for estrogen biosynthesis) expression and E(2) release. Additionally, TGF-β3 enhanced the binding of SF-1 to endogenous ovary-specific Cyp19a1 type II promoter, as evidenced by chromatin immunoprecipitation assays. The enhanced effect of SF-1 by TGF-β3 may be mediated through functional interactions between SF-1 and mothers against decapentaplegic homolog (Smad)3 (a mediator of TGF-β signaling pathway), because disruption of the interaction abolished the synergistic effects of SF-1, Smad3, and TGF-β3 on Cyp19a1 mRNA expression. RNA interference and chromatin immunoprecipitation studies also demonstrated that Smad3 was required for SF-1 binding to Cyp19a1 type II promoter and activation of Cyp19a1. Smad3 thus acts as a point of convergence that involves integration of SF-1 and TGF-β signaling in affecting E(2) production. Taken together, our data provide mechanistic insights into the roles of SF-1 in TGF-β3-mediated E(2) synthesis. Understanding of potential cross-points between extracellular signals affecting estrogen production will help to discover new therapeutic targets in estrogen-related diseases.
[本文引用: 2]
URLPMID:17192407 [本文引用: 1]
Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17beta-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17beta-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.
URL [本文引用: 1]
目的 转录因子FOXL2是颗粒细胞雌激素合成与颗粒细胞功能维持的关键调控分子,但目前尚不清楚FOXL2蛋白的表达及功能调控机制.文章旨在鉴定调控FOXL2蛋白功能的新分子.方法 通过分离小鼠卵巢组织进行免疫组化染色检测DNAJB11的表达定位情况,含有全长DNAJB11基因cDNA序列(Ad-DNAJB11-Flag和Ad-Flag-DNAJB11)的腺病毒载体按AdMax系统(Microbix)方法进行病毒颗粒的包装.按NE-PERTM Nuclear and Cytoplasmic抽提试剂盒所述方法进行细胞核质蛋白的分离.KGN细胞感染不同浓度腺病毒(Ad-DNAJB11和Ad-LacZ),测量波长450 nm的吸光度值,进行细胞增殖检测.采用Access Immunoassay System 2化学发光自动检测系统(Beckman Coulter)检测细胞培养上清中雌二醇的浓度.采用PCR方法扩增含有人FOXL2和人DNAJB 11基因的全长cDNA序列,并分别亚克隆至pCS2-6XMT载体(Myc-FOXL2)和pFLAG-CMV2载体(Flag-DNAJB11)中.采用Lipofectamine 2000转染试剂将Myc-FOXL2和Flag-DNAJB11表达质粒瞬时转染至细胞HEK-293细胞中.收集HEK-293细胞或FSH处理的KGN细胞的裂解液,进行FOXL2和DNAJB11的免疫共沉淀实验.通过细胞免疫荧光染色和荧光素酶报告基因等实验检测内质网Hsp40/DnaJ家族成员DNAJB11调控颗粒细胞的雌激素水平.结果 Western blot表明DNAJB11蛋白在人卵癌颗粒细胞KGN、小鼠原代卵巢颗粒细胞mGC以及小鼠卵巢中高表达,免疫组化染色结果亦证实DNAJB11蛋白表达于小鼠卵巢的卵母细胞质中,并在窦前卵泡、排卵前卵泡的颗粒细胞中持续表达.免疫荧光染色和Western blot分析亦表明外源性激素FSH可以诱导KGN细胞中内源性DNAJB11蛋白从内质网转移到细胞核中.腺病毒介导的DNAJB11-Flag过表达定位于细胞内质网中,但当Flag标签阻断DNAJB11信号肽功能时,外源Flag-DNAJB11过表达则定位于颗粒细胞的细胞核,内质网中DNAJB11-Flag蛋白高表达以浓度依赖的方式减少KGN细胞中雌二醇的合成,与Ad-LacZ(MOI=50)比较,Ad-DNAJB11-Flag(MOI=50)减少,KGN细胞中雌二醇的合成[(10749.0±801.7)pg/mL vs(79030.±409.5)pg/mL,P<0.01],而细胞核中高表达Flag-DNAJB11后则刺激KGN细胞中雌二醇的生成,与Ad-LacZ(MOI=50)比较,Ad-DNAJB11-Flag(MOI=50)刺激雌二醇的生成[(10749.0±801.7)pg/mL vs(14217.0±1218.0)pg/mL,P<0.01].免疫共沉淀实验表明当Myc-tagged FOXL2与Flag-tagged DNAJB11分别共转染HEK 293细胞,FOXL2与DNAJB11可以相互免疫共沉淀对方,荧光素酶报告基因实验进一步表明细胞核中表达的DNAJB11显著增强FOXL2介导的Cyp19A1启动子活性和Cyp19A1蛋白表达.结论 DNAJB11是转录因子FOXL2新的结合分子并调控FOXL2蛋白的稳定性与转录活性.
URL [本文引用: 1]
目的 转录因子FOXL2是颗粒细胞雌激素合成与颗粒细胞功能维持的关键调控分子,但目前尚不清楚FOXL2蛋白的表达及功能调控机制.文章旨在鉴定调控FOXL2蛋白功能的新分子.方法 通过分离小鼠卵巢组织进行免疫组化染色检测DNAJB11的表达定位情况,含有全长DNAJB11基因cDNA序列(Ad-DNAJB11-Flag和Ad-Flag-DNAJB11)的腺病毒载体按AdMax系统(Microbix)方法进行病毒颗粒的包装.按NE-PERTM Nuclear and Cytoplasmic抽提试剂盒所述方法进行细胞核质蛋白的分离.KGN细胞感染不同浓度腺病毒(Ad-DNAJB11和Ad-LacZ),测量波长450 nm的吸光度值,进行细胞增殖检测.采用Access Immunoassay System 2化学发光自动检测系统(Beckman Coulter)检测细胞培养上清中雌二醇的浓度.采用PCR方法扩增含有人FOXL2和人DNAJB 11基因的全长cDNA序列,并分别亚克隆至pCS2-6XMT载体(Myc-FOXL2)和pFLAG-CMV2载体(Flag-DNAJB11)中.采用Lipofectamine 2000转染试剂将Myc-FOXL2和Flag-DNAJB11表达质粒瞬时转染至细胞HEK-293细胞中.收集HEK-293细胞或FSH处理的KGN细胞的裂解液,进行FOXL2和DNAJB11的免疫共沉淀实验.通过细胞免疫荧光染色和荧光素酶报告基因等实验检测内质网Hsp40/DnaJ家族成员DNAJB11调控颗粒细胞的雌激素水平.结果 Western blot表明DNAJB11蛋白在人卵癌颗粒细胞KGN、小鼠原代卵巢颗粒细胞mGC以及小鼠卵巢中高表达,免疫组化染色结果亦证实DNAJB11蛋白表达于小鼠卵巢的卵母细胞质中,并在窦前卵泡、排卵前卵泡的颗粒细胞中持续表达.免疫荧光染色和Western blot分析亦表明外源性激素FSH可以诱导KGN细胞中内源性DNAJB11蛋白从内质网转移到细胞核中.腺病毒介导的DNAJB11-Flag过表达定位于细胞内质网中,但当Flag标签阻断DNAJB11信号肽功能时,外源Flag-DNAJB11过表达则定位于颗粒细胞的细胞核,内质网中DNAJB11-Flag蛋白高表达以浓度依赖的方式减少KGN细胞中雌二醇的合成,与Ad-LacZ(MOI=50)比较,Ad-DNAJB11-Flag(MOI=50)减少,KGN细胞中雌二醇的合成[(10749.0±801.7)pg/mL vs(79030.±409.5)pg/mL,P<0.01],而细胞核中高表达Flag-DNAJB11后则刺激KGN细胞中雌二醇的生成,与Ad-LacZ(MOI=50)比较,Ad-DNAJB11-Flag(MOI=50)刺激雌二醇的生成[(10749.0±801.7)pg/mL vs(14217.0±1218.0)pg/mL,P<0.01].免疫共沉淀实验表明当Myc-tagged FOXL2与Flag-tagged DNAJB11分别共转染HEK 293细胞,FOXL2与DNAJB11可以相互免疫共沉淀对方,荧光素酶报告基因实验进一步表明细胞核中表达的DNAJB11显著增强FOXL2介导的Cyp19A1启动子活性和Cyp19A1蛋白表达.结论 DNAJB11是转录因子FOXL2新的结合分子并调控FOXL2蛋白的稳定性与转录活性.
URL [本文引用: 1]
URLPMID:24174573 [本文引用: 1]
We previously established a potential role for cocaine and amphetamine regulated transcript (CARTPT) in dominant follicle selection in cattle. CARTPT expression is elevated in subordinate versus dominant follicles, and treatment with the mature form of the CARTPT peptide (CART) decreases follicle-stimulating hormone (FSH)-stimulated granulosa cell estradiol production in vitro and follicular fluid estradiol and granulosa cell CYP19A1 mRNA in vivo. However, mechanisms that regulate granulosa cell CART responsiveness are not understood. In this study, we investigated hormonal regulation of granulosa cell CART-binding sites in vitro and temporal regulation of granulosa cell CART-binding sites in bovine follicles collected at specific stages of a follicular wave. We also determined the effect of inhibition of CART receptor signaling in vivo on estradiol production in future subordinate follicles. Granulosa cell CART binding in vitro was increased by FSH, and this induction was blocked by estrogen receptor antagonist treatment. In follicles collected in vivo at specific stages of a follicular wave, granulosa cell CART binding in the F2 (second largest), future subordinate follicle increased during dominant follicle selection. Injection into the F2 follicle (at onset of diameter deviation) of an inhibitor of the o/i subclass of G proteins (previously shown to block CART actions in vitro) resulted in increased follicular fluid estradiol concentrations in vivo. Collectively, results demonstrate hormonal regulation of granulosa cell CART binding in vitro and temporal regulation of CART binding in subordinate follicles during dominant follicle selection. Results also suggest that CART signaling may help suppress estradiol-producing capacity of the F2 (subordinate) follicle during this time period.
URL [本文引用: 1]
目的 探讨多囊卵巢综合征中结缔组织生长因子(CTGF)的表达及对卵巢颗粒细胞增殖凋亡的影响及机制.方法 60只SD大鼠随机分为正常组和实验组,每组各30只.实验组大鼠肌肉注射脱氢表雄酮(DHEA)(6 mg/100 g体重)和0.2 ml的注射用油,正常组大鼠肌肉注射0.2 ml的注射用油.摘取多囊卵巢综合征大鼠模型的卵巢组织,提取组织中的总RNA和总蛋白,Western blot检测CTGF的mRNA和蛋白表达;原代分离培养卵巢颗粒细胞,将NC-siRNA、CTGF-siRNA转染至细胞内,不经任何处理的细胞作为对照组.48 h后CCK8实验和流式细胞仪分别检测细胞的增殖及凋亡情况;Western blot检测Cleaved caspase3、PI3K、p-AKT蛋白表达.结果 卵巢组织中CTGF的mRNA和蛋白表达均显著高于正常组(P<0.01);转染siRNA后能显著降低CTGF的蛋白表达;CTGF-siRNA组细胞存活率及PI3K、p-AKT蛋白表达显著低于对照组和NC-siRNA组,细胞凋亡率及Cleaved caspase3蛋白表达显著高于对照组和NC-siRNA组(P<0.01).结论 CTGF在多囊卵巢综合征中的表达升高,抑制其表达可降低颗粒细胞的增殖及诱导凋亡,其机制与PI3K/AKT信号通路的调控有关.
URL [本文引用: 1]
目的 探讨多囊卵巢综合征中结缔组织生长因子(CTGF)的表达及对卵巢颗粒细胞增殖凋亡的影响及机制.方法 60只SD大鼠随机分为正常组和实验组,每组各30只.实验组大鼠肌肉注射脱氢表雄酮(DHEA)(6 mg/100 g体重)和0.2 ml的注射用油,正常组大鼠肌肉注射0.2 ml的注射用油.摘取多囊卵巢综合征大鼠模型的卵巢组织,提取组织中的总RNA和总蛋白,Western blot检测CTGF的mRNA和蛋白表达;原代分离培养卵巢颗粒细胞,将NC-siRNA、CTGF-siRNA转染至细胞内,不经任何处理的细胞作为对照组.48 h后CCK8实验和流式细胞仪分别检测细胞的增殖及凋亡情况;Western blot检测Cleaved caspase3、PI3K、p-AKT蛋白表达.结果 卵巢组织中CTGF的mRNA和蛋白表达均显著高于正常组(P<0.01);转染siRNA后能显著降低CTGF的蛋白表达;CTGF-siRNA组细胞存活率及PI3K、p-AKT蛋白表达显著低于对照组和NC-siRNA组,细胞凋亡率及Cleaved caspase3蛋白表达显著高于对照组和NC-siRNA组(P<0.01).结论 CTGF在多囊卵巢综合征中的表达升高,抑制其表达可降低颗粒细胞的增殖及诱导凋亡,其机制与PI3K/AKT信号通路的调控有关.
URLPMID:23045700 [本文引用: 2]
Abstract Controlled maturation of ovarian follicles is necessary for fertility. Follicles are restrained at an immature stage until stimulated by FSH secreted by pituitary gonadotropes. FSH acts on granulosa cells within the immature follicle to inhibit apoptosis, promote proliferation, stimulate production of steroid and protein hormones, and induce ligand receptors and signaling intermediates. The phosphoinositide 3-kinase (PI3K)/AKT (protein kinase B) pathway is a pivotal signaling corridor necessary for transducing the FSH signal. We report that protein kinase A (PKA) mediates the actions of FSH by signaling through multiple targets to activate PI3K/AKT. PKA uses a route that promotes phosphorylation of insulin receptor substrate-1 (IRS-1) on Tyr(989), a canonical binding site for the 85-kDa regulatory subunit of PI3K that allosterically activates the catalytic subunit. PI3K activation leads to activation of AKT through phosphorylation of AKT on Thr(308) and Ser(473). The adaptor growth factor receptor bound protein 2-associated binding protein 2 (GAB2) is present in a preformed complex with PI3K heterodimer and IRS-1, it is an A-kinase anchoring protein that binds the type I regulatory subunit of PKA, and it is phosphorylated by PKA on Ser(159). Overexpression of GAB2 enhances FSH-stimulated AKT phosphorylation. GAB2, thus, seems to coordinate signals from the FSH-stimulated rise in cAMP that leads to activation of PI3K/AKT. The ability of PKA to commandeer IRS-1 and GAB2, adaptors that normally integrate receptor/nonreceptor tyrosine kinase signaling into PI3K/AKT, reveals a previously unrecognized route for PKA to activate a pathway that promotes proliferation, inhibits apoptosis, enhances translation, and initiates differentiation of granulosa cells.
URL [本文引用: 1]
URLPMID:29396446 [本文引用: 3]
Sma-and Mad-related protein 4 (SMAD4) is the central mediator of the transforming growth factor beta signaling pathway and is closely related to mammalian reproductive ability and the development of ovarian follicles. However, little is currently known about the role of SMAD4 in mammalian follicular granulosa cell (GC) apoptosis or its regulation by miRNAs. Here, we found that the porcine... [Show full abstract]
[本文引用: 3]
URLPMID:29319157 [本文引用: 1]
Abstract SMAD7 disrupts the TGF-β signaling pathway by influencing TGFBR1 stability and by blocking the binding of TGFBR1 to SMAD2/3. In this study, we showed that SMAD7 attenuated the TGF-β signaling pathway in ovarian granulosa cells (GCs) by regulating TGFBR1 transcriptional activity. To function as a transcription factor, SMAD7 downregulated the mRNA levels of TGFBR1 via direct binding to the SMAD-binding elements (SBEs) within the promoter region of pig TGFBR1. We also showed that SMAD7 enhanced porcine GC apoptosis by interrupting TGFBR1 and the TGF-β signaling pathway. Interestingly, miR-181b, a microRNA that is downregulated during porcine follicular atresia, was identified to be directly targeting SMAD7 at its 3'-UTR. By inhibiting SMAD7, miR-181b could inhibit GC apoptosis by activating the TGF-β signaling pathway. Our findings provide new insights into the mechanisms underlying the regulation of the TGF-β signaling pathway by SMAD7 and miR-181b. This article is protected by copyright. All rights reserved
URLPMID:25448599 [本文引用: 2]
Smad7 has a key role in apoptosis of mammalian ovarian granulosa cells (GCs), as it antagonizes and fine-tunes transforming growth factor 尾 (TGF尾) signaling. This study demonstrates that miR-92a regulates GC apoptosis in pig ovaries by targeting Smad7 directly. The expression level of miR-92a was down-regulated in atretic porcine follicles, whereas miR-92a expression led to inhibition of GC apoptosis. The Smad7 gene was identified as a direct target of miR-92a using a dual-luciferase reporter assay. Transfection of GCs with miR-92a mimics decreased Smad7 mRNA and protein levels, whereas expression of an miR-92a inhibitor in GCs had the opposite effect. In addition, knockdown of Smad7 prevented GC apoptosis in cells that expressed the miR-92a inhibitor.
URLPMID:4736824 [本文引用: 1]
Abstract The Notch pathway is an essential regulator of cell proliferation and differentiation during development. Its involvement in insect oogenesis has been examined in insect species with meroistic ovaries, and it is known to play a fundamental role in cell fate decisions and the induction of the mitosis-to-endocycle switch in follicular cells (FCs). This work reports the functions of the main components of the Notch pathway (Notch and its ligands Delta and Serrate) during oogenesis in Blattella germanica, a phylogenetically basal species with panoistic ovary. As is revealed by RNAi-based analyses, Notch and Delta were found to contribute towards maintaining the FCs in an immature, non-apoptotic state. This ancestral function of Notch appears in opposition to the induction of transition from mitosis to endocycle that Notch exerts in Drosophila melanogaster, a change in the Notch function that might be in agreement with the evolution of the insect ovary types. Notch was also shown to play an active role in inducing ovarian follicle elongation via the regulation of the cytoskeleton. In addition, Delta and Notch interactions were seen to determine the differentiation of the posterior population of FCs. Serrate levels were found to be Notch-dependent and are involved in the control of the FC programme, although they would appear to play no crucial role in panoistic ovary oogenesis. 2016 The Authors.
URLPMID:18818300 [本文引用: 1]
Notch signaling directs cell fate during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating folliculogenesis are beginning to emerge from mouse genetic models. We investigated how Notch signaling might influence the formation of primordial follicles. Follicle assembly takes place when germ cell syncytia within the ovary break down and germ cells are encapsulated by pregranulosa cells. In the mouse, this occurs during the first 4-5 d of postnatal life. The expression of Notch family genes in the neonatal mouse ovary was determined through RT-PCR measurements. Jagged1, Notch2, and Hes1 transcripts were the most abundantly expressed ligand, receptor, and target gene, respectively. Jagged1 and Hey2 mRNAs were up-regulated over the period of follicle formation. Localization studies demonstrated that JAGGED1 is expressed in germ cells prior to follicle assembly and in the oocytes of primordial follicles. Pregranulosa cells that surround germ cell nests express HES1. In addition, pregranulosa cells of primordial follicles expressed NOTCH2 and Hey2 mRNA. We used an ex vivo ovary culture system to assess the requirement for Notch signaling during early follicle development. Newborn ovaries cultured in the presence of gamma-secretase inhibitors, compounds that attenuate Notch signaling, had a marked reduction in primordial follicles compared with vehicle-treated ovaries, and there was a corresponding increase in germ cells that remained within nests. These data support a functional role for Notch signaling in regulating primordial follicle formation.
URLPMID:28400072 [本文引用: 1]
Abstract The Notch signaling pathway regulates cell proliferation, differentiation and apoptosis involved in development of the organs and tissues such as nervous system, cartilage, lungs, kidneys and prostate as well as the ovarian follicles. This study aimed to investigate the mRNA expression and localization of NOTCH2, as the key factor in Notch signaling pathway. This was determined by PCR, real-time PCR and immunohistochemistry. Additionally, the effects of inhibiting Notch signaling pathway with different concentrations (5渭M, 10渭M and 20渭M) of N-[N-(3, 5-Difuorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling pathway, on ovine granulosa cells was determined in vitro by detecting estradiol production using enzyme linked immunosorbent assay and expressions of the genes related to the cell cycle and apoptosis using real-time polymerase chain reaction (PCR). NOTCH2, the key member of Notch signaling pathway, was found in ovine follicles, and the expression of NOTCH2 mRNA was highest in the theca cells of the follicles in medium sizes (3-5mm in diameter) and granulosa cells of the follicles in large sizes (>5mm in diameter). Immunohistochemical results demonstrated that NOTCH2 protein was expressed in granulosa cells of preantral follicles, in both granulosa cells and theca cells of antral follicles. Compared with DAPT-treated groups, the control group had a higher number of granulosa cells (P<0.05) and a higher estradiol production (P<0.05). Compared with the control group, the mRNA abundances of HES1, MYC, BAX, BCL2 and CYP19A1 in DAPT-treated groups was lower (P<0.05), respectively; whereas, the expression of CCND2, CDKN1A and TP53 mRNA showed no remarkable difference compared with control group. Collectively, Notch signaling pathway could be involved in the ovine follicular development by regulating the growth and estradiol production of granulosa cells. Copyright 2017 Elsevier B.V. All rights reserved.
URLPMID:29126263 [本文引用: 1]
Notch signaling directs cell fates during embryogenesis by influencing cell proliferation, differentiation, and apoptosis. Notch genes are expressed in the adult mouse ovary, and roles for Notch in regulating ovarian follicle maturation are beginning to emerge from mouse genetic models. We are investigating how Notch signaling might influence the formation of primordial follicles. Our studies... [Show full abstract]
URLPMID:27833138 [本文引用: 4]
Broodiness, a maternal behavior and instinct for natural breeding in poultry, inhibits egg production and affects the poultry industry. Phenotypic and physiological factors influencing broodiness in poultry have been extensively studied, but the molecular regulation mechanism of broodiness remains unclear. Effective research strategies focusing on broodiness are hindered by limited understanding of goose developmental biology. Here we established the transcriptomes of goose follicles at egg-laying and broody stages by Illumina HiSeq platform and compared the sequenced transcriptomes of three types of follicles (small white, large white and small yellow). It was found that there were 92 up-regulated and 84 down-regulated transcription factors and 101 up-regulated and 51 down-regulated hormone-related genes. Many of these genes code for proteins involved in hormone response, follicular development, autophagy, and oxidation. Moreover, the contents of progesterone and estradiol in follicles were altered, and the autophagy levels of follicles were enhanced during the broody stage. These results suggest that hormone- and autophagy-signaling pathways are critical for controlling broodiness in the goose. We demonstrated that transcriptome analysis of egg-laying and broody Zhedong white goose follicles provided novel insights into broodiness in birds.
