,1, 狄冉1, 刘秋月1, 胡文萍1, 王翔宇1, 田志龙1, 张效生2, 张金龙2, 储明星,1Expression Analysis of Five Genes in the Gonadal Axis of Small Tail Han Sheep and Sunite Sheep
ZHANG ZhuangBiao,1, DI Ran1, LIU QiuYue1, HU WenPing1, WANG XiangYu1, TIAN ZhiLong1, ZHANG XiaoSheng2, ZHANG JinLong2, CHU MingXing,1通讯作者:
第一联系人:
收稿日期:2018-04-18接受日期:2018-07-7网络出版日期:2018-12-26
| 基金资助: |
Received:2018-04-18Accepted:2018-07-7Online:2018-12-26
摘要
关键词：
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本文引用格式
张壮彪, 狄冉, 刘秋月, 胡文萍, 王翔宇, 田志龙, 张效生, 张金龙, 储明星. 5个基因在小尾寒羊和苏尼特羊性腺轴相关组织中表达分析[J]. 中国农业科学, 2018, 51(24): 4710-4719 doi:10.3864/j.issn.0578-1752.2018.24.011
ZHANG ZhuangBiao, DI Ran, LIU QiuYue, HU WenPing, WANG XiangYu, TIAN ZhiLong, ZHANG XiaoSheng, ZHANG JinLong, CHU MingXing.
0 引言
【研究意义】对于绵羊来说,排卵数[1]和产羔数是衡量繁殖力高低的两个重要因素。不同物种之间的后代数和排卵数差别很大,比如人和牛一般被认为是胎产单个后代的物种,而小鼠和猪一般被看作窝产多仔的物种。绵羊排卵数一般仅有1—2个,但其有较大的“可塑性”。有研究表明某些羊的排卵数可达10个左右[2]。绵羊繁殖力受营养、羊群公母比例等多种因素调控,其中遗传因素对绵羊的繁殖力影响较大。如FecB的发现,携带该基因的母羊在排卵数方面存在剂量效应,即每增加一个拷贝数其排卵数可增加1.5个,产羔数可增加1—1.5个左右[3,4],该基因的发现为选育高繁殖力母羊提供了科学依据。但近年来随着研究不断深入,研究人员发现还有一些其他基因比如骨形态发生蛋白2、6、7（bone morphogenetic protein 2、6、7, BMP2、6、7）、钙蛋白酶抑制蛋白（calpastatin,CAST）和可卡因-苯丙胺调节转录肽（cocaine and amphetamine regulated transcript,CART）[1, 5-7]可以影响绵羊的繁殖力。探究这些基因在单羔、多羔羊性腺轴相关组织中的表达差异对于阐明相关分子机理具有重要意义。【前人研究进展】BMPs家族属于转化生子因子亚家族的成员[8],其发挥作用主要是通过BMP II型受体（BMPR-II）以及BMPR IA或IB型受体形成异低聚物的形式发挥信号转导作用。这种复合体会使II型受体磷酸化I型受体,进而导致Smad蛋白磷酸化,形成异二聚体之后导致靶基因转录改变[9,10],从而影响母羊的繁殖力。该家族对哺乳动物繁殖力影响首先是在1999年由SHIMASAKI等[11]报道的。研究表明在哺乳动物生殖系统中,组织特异性表达的BMPs家族成员能够影响某些结构和器官的形成以及其生物学功能的发挥[10]。LOCHAB 等[12]发现在原始生殖细胞向配子发育的过程中BMPs家族起主要调控作用。在动物颗粒细胞中它主要是通过调控促性腺激素诱导的生成类固醇以及有丝分裂发挥其调控作用[13]。研究表明BMPs家族成员能明显促进牛、羊的卵泡发育[5,14-16],也有研究发现该家族成员与闭锁卵泡[17]有一定的关系。CCS (calpain-calpastatin system)是机体内重要的系统之一,该系统对包括T细胞[18]等多种细胞或系统具有一定的调控作用。该系统是由钙蛋白酶（calpain）（m和μ型钙蛋白）和CAST组成。作为该系统的重要成员,CAST基因编码钙蛋白酶抑制蛋白,它能专一性地识别相应蛋白酶与钙离子结合后构像的变化,从而发挥蛋白酶抑制剂的调控作用。研究发现它对一些产肉性状比如肌肉形成、初生重、断奶重以及后期肉品质的质量起重要作用[7,19-21]。该基因对绵羊繁殖性能[7,21]以及牛卵泡封闭[22]也有一定影响。前人研究已经证明脑垂体中的性腺激素对于卵巢的卵泡发育和排卵具有重要作用[23,24],故之前研究的关注点是与繁殖相关的性腺激素。但是随着科技的进步和人们对繁殖机理研究的不断深入,越来越多的证据证明对排卵数有明显影响的卵巢局部调节因子[25,26,27]。CART作为一种卵巢的局部调节因子在2004年由KOBAYASHI等首先发现[28,29],当时研究人员发现它是一种与体增重和某些神经反应有关的神经因子的存在[30]。该基因对牛、羊体内的某些激素如雌激素具有一定的抑制作用[31,32],对于牛卵巢、卵泡的发育以及卵泡的闭锁具有重要影响[33,34,35]。该基因在动物采食活动、液体平衡、感官刺激传导以及免疫反应[36,37,38]中也发挥重要作用。【本研究切入点】BMPs家族、CAST、CART对绵羊繁殖具有一定的调控作用,目前关于这些基因在苏尼特羊、小尾寒羊性腺轴相关组织中表达情况还没有文献详细报道。【拟解决的关键问题】故本文选用BMPs家族中具有代表性的BMP2、BMP6、BMP7以及CAST、CART作为研究对象,以期通过表达谱的方式说明其在单羔、多羔绵羊性腺轴各组织表达量的差异情况,为进一步阐明上述5个基因影响绵羊繁殖力的分子机理提供一定的参考,为“分子编写育种”[39]提供一定的遗传学材料。1 材料与方法
1.1 试验时间、地点
本研究室内试验于2017年12月至2018年3月在中国农业科学院北京畜牧兽医研究所肉羊遗传育种科研团队实验室进行。1.2 实验样品的采集和保存
随机选取2—3周岁健康、经产的空怀苏尼特母羊和小尾寒羊母羊各3只,于2017年10月饲养于天津市畜牧兽医研究所试验羊场,所有试验羊的饲养环境和饲料均相同。2017年11月进行屠宰,取其大脑、小脑、下丘脑、垂体、子宫、卵巢、输卵管,取样后迅速装入1.8mL RNase-Free冻存管中（最大样品量为冻存管体积的2/3）。所有样品采集要在半小时内完成,样品采完后迅速放入液氮中冷冻保存,用干冰带回实验室,放入-80℃冰箱冷冻保存,备用。1.3 RNA的提取及质量检测
将采集的小尾寒羊和苏尼特羊性腺轴7种相关组织研磨后,用Trizol（Invitrogen,美国）进行裂解,之后按照动物组织RNA提取试剂盒（天根,北京）的说明进行总RNA的提取,用1.0%琼脂糖凝胶电泳和NANODROP 2000检测提取RNA的质量和浓度。经检验合格的组织总RNA置于-80℃保存备用。1.4 引物设计
参考文献中已报道的BMP2[40]、BMP7[40]、BMP6[41]、CART[1]基因序列并结合Primer Premier 5.0进行引物的合成（NCBI的登录号分别为XM_ 004014353.3、NM_001308564.1、XM_015102858.1、XM_015101190.1）。根据GenBank提供的绵羊CAST、β-actin基因序列（登录号分别为NM_001009788.1、NM_001009784）信息并用Primer Premier 5.0软件进行引物设计,其中β-actin作为内参基因。引物由北京天一辉远生物科技有限公司合成。各引物浓度均为10 μmol·L-1,其他详细信息见表1。Table 1
表1
表1引物的序列、扩增片段大小及退火温度
Table 1
| 基因名称 Name | 引物序列 Primer sequence | 片段大小 Length (bp) | 退火温度 Tm (℃) |
|---|---|---|---|
| BMP2 | F:5′-ATCACCTGAACTCCACGAA-3′ | 140 | 60 |
| R: 5′-TACCACCTTCTCATTCTCATC-3′ | |||
| BMP6 | F:5′- AGCAGACTACAACAGCAGCGA-3′ | 267 | 60 |
| R:5′- AGCACCGAGATGGCGTTCA-3′ | |||
| BMP7 | F:5′- AAAACAGCAGCAGCGACCAGAG-3′ | 123 | 68 |
| R:5′- CCTCACAGTAGTAGGCGGCATAGC-3′ | |||
| CAST | F:5′-TGGGGCCCAATGACGCCATCGATG - 3′ | 222 | 60 |
| R:5′- GGTGGAGCAGCACTTCTGATCACC - 3′ | |||
| CART | F:5′- AGCAGCCACCATCAGAGAAA - 3′ | 214 | 60 |
| R:5′- TCCAGAGCAGTATCCATGTCT - 3′ | |||
| β-actin | F:5′- CCAACCGTGAGAAGATGACC-3′ | 97 | 60 |
| R:5′- CCCGAGGCGTACAGGGACAG-3′ |
新窗口打开|下载CSV
1.5 cDNA的合成
利用反转录试剂盒合成cDNA,反转录体系和反应条件见表2,全程在冰上操作。将反转录完成之后的cDNA产物稀释后,用持家基因β-actin进行PCR检测,质量合格后-20℃保存以备检测基因mRNA表达。Table 2
表2
表2反转录体系
Table 2
| 试剂 Reagent | 各试剂加入量 Volume of each reagent |
|---|---|
| PrimeScript 反转录混合引物Ⅰ PrimeScript RT Enzyme Mix Ⅰ | 1.0 μL |
| Oligo dT引物 Oligo dT Primer | 1.0 μL |
| 随机引物 Random 6 mers | 1.0 μL |
| 5×PrimeScript荧光定量缓冲液 5×PrimeScript Buffer (for Real Time) | 4.0 μL |
| RNA | 1.0 μg |
| 双蒸水 RNase-Free ddH2O | 12μL |
新窗口打开|下载CSV
1.6 实时荧光定量PCR
1.6.1 实时荧光定量PCR体系和程序 反应体系总体积为20 μL,具体各试剂用量见表3。PCR程序：95℃预变性5min ,95℃变性5 s,60℃ 30 s,40个循环;反应结束后进行熔解曲线分析。Table 3
表3
表3实时荧光定量PCR体系
Table 3
| 试剂 Reagent | 各试剂加入量 Volume of each reagent (μL) |
|---|---|
| 荧光定量Ex Taq引物Ⅱ SYBR Premix Ex TaqⅡ | 10.0 |
| 上游引物（10 μmol·L-1） Upstream primer (10 μmol·L-1) | 0.8 |
| 下游引物（10 μmol·L-1） Downstream primer (10 μmol·L-1) | 0.8 |
| cDNA 模板（500 ng·μL-1） cDNA template (500 ng·μL-1) | 2.0 |
| 双蒸水 RNase-Free ddH2O | 6.4 |
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1.6.2 标准曲线的建立 将cDNA样本5倍稀释后,进行2倍梯度稀释获得5个浓度梯度（1、1/2、1/4、1/8、1/16）的cDNA样品。用这些cDNA作为模板对目的基因和持家基因进行荧光定量PCR,以浓度梯度的对数值（10为底数）为横坐标,以检测所得Ct值为纵坐标,绘制目的基因和持家基因标准曲线。
1.6.3 实时荧光定量检测与数据分析 使用Roche Light Cycler?480Ⅱ型荧光定量PCR仪进行荧光定量检测,采用Excel进行数据处理,用2-△△Ct的方法计算基因的相对表达量。获得的表达量数据用SPSS19.0进行单因素方差分析（One-way ANOVA）,分为差异显著（P＜0.05）;差异极显著（P＜0.01）。
2 结果
2.1 RNA的提取
提取后的总RNA用1%琼脂糖凝胶电泳进行检测,其完整性如图1。结果发现28S条带明显亮于18S条带,条带完整性较好,表明该RNA质量合格,可用于后续试验。图1
新窗口打开|下载原图ZIP|生成PPT图1RNA电泳检测
Fig. 1Electrophoresis of the RNA
2.2 BMP2、BMP6、BMP7、CAST、CART组织表达分析
2.2.1 BMP2组织表达分析 从图2中可以看出该基因在7种组织中均有表达,其中在小尾寒羊的输卵管表达量最高;在小尾寒羊输卵管、卵巢、垂体和小脑该基因的表达量极显著高于苏尼特羊（P＜0.01）;该基因在小尾寒羊下丘脑的表达量显著高于苏尼特羊（P＜0.05）;其他组织差异均不显著（P＞0.05）。图2
新窗口打开|下载原图ZIP|生成PPT图2BMP2在小尾寒羊和苏尼特羊各组织中的表达
Fig. 2Expression of BMP2 in tissues of Small Tail Han sheep and Sunite sheep
2.2.2 BMP6组织表达分析 由图3可见,在两种绵羊的7种组织中,该基因在小尾寒羊的卵巢中表达量最高;该基因在小尾寒羊卵巢、输卵管、垂体的表达量极显著高于苏尼特羊（P＜0.01）;该基因在苏尼特羊垂体和卵巢表达量很少或基本不表达,其他组织差异都不显著（P＞0.05）。
图3
新窗口打开|下载原图ZIP|生成PPT图3BMP6在小尾寒羊和苏尼特羊各组织中的表达
Fig. 3Expression of BMP6 in tissues of Small Tail Han and Sunite sheep
2.2.3 BMP7基因组织表达分析 如图4所示,该基因在小尾寒羊垂体中表达量最高;该基因在小尾寒羊下丘脑、输卵管和卵巢中的表达量极显著高于苏尼特羊（P＜0.01）;在小尾寒羊垂体中该基因的表达量显著高于苏尼特羊（P＜0.05）,在苏尼特羊大脑中该基因的表达量显著高于小尾寒羊（P＜0.05）;其他组织差异均不显著（P＞0.05）。
图4
新窗口打开|下载原图ZIP|生成PPT图4BMP7在小尾寒羊和苏尼特羊各组织中的表达
Fig. 4Expression of BMP7 in tissues of Small Tail Han and Sunite sheep
2.2.4 CAST组织表达分析 如图5所示,CAST在小尾寒羊输卵管和子宫中的表达量极显著高于苏尼特羊（P＜0.01）;在苏尼特羊小脑中该基因的表达量显著高于小尾寒羊（P＜0.05）;而该基因在两种绵羊的下丘脑、垂体中均呈痕量表达。
图5
新窗口打开|下载原图ZIP|生成PPT图5CAST在小尾寒羊和苏尼特羊各组织中的表达
Fig. 5Expression of CAST in tissues of Small Tail Han and Sunite sheep
2.2.5 CART组织表达分析 如图6所示,该基因在两种绵羊下丘脑表达量最高;该基因在小尾寒羊大脑中表达量极显著高于苏尼特羊（P＜0.01）;在苏尼特羊垂体中该基因的表达量显著高于小尾寒羊（P＜0.05）;该基因在小尾寒羊和苏尼特羊卵巢、子宫和输卵管均呈痕量表达。
图6
新窗口打开|下载原图ZIP|生成PPT图6CART在小尾寒羊和苏尼特羊各组织中的表达
Fig. 6Expression of CART in tissues of Small Tail Han and Sunite sheep
3 讨论
3.1 BMPs家族对繁殖的调控
卵巢、子宫等是动物最重要的繁殖器官,探究影响繁殖相关基因在这些组织中的表达的差异对于阐明产羔数差异和繁殖力的高低具有一定的促进作用。研究表明不同的BMPs家族成员在不同的物种体内均有表达[8,42-46],说明该基因家族可能在动物体内发挥着重要调控作用。有研究表明在绵羊卵巢的卵母细胞、颗粒细胞、膜细胞均存在BMPs受体,说明BMPs家族可能会在卵巢发挥其局部调节因子的作用,从而影响绵羊的繁殖活动[4,47]。邝美倩等[48]研究表明BMP2在产羔数较高的湖羊的子宫中有较高表达量。在本实验中,BMP2不仅在产多羔的小尾寒羊的子宫有一定的表达量,而且在苏尼特羊也有一定的表达量,但二者差异不显著（P＞0.05）。徐业芬等[40]在湖羊的相关组织表达谱中发现BMP6在卵巢和输卵管有表达,但在子宫中不表达;BMP2、BMP7在卵巢组织中有表达,但是上述3个基因在单、双羔湖羊的卵巢表达差异并不显著（P＞0.05）。本研究显示BMP6不仅在卵巢和输卵管中有表达,而且在小尾寒羊（多羔）和苏尼特羊（单羔）的子宫中均有表达;并且3个基因在小尾寒羊（多羔）卵巢中均有表达且表达量极显著高于苏尼特羊（单羔）（P＜0.01）。也有研究发现当卵巢发生某些病变如肿性卵巢疾病时,BMPs家族的BMP2、BMP6等的表达量会发生改变[49],所以说某些卵巢的病变也有可能是导致其基因表达量差异的重要原因。在动物的繁殖周期中,激素是一类非常重要的调控发情或繁殖的因素。BMPs家族基因对繁殖相关的激素具有重要调控作用。LIU等[50]研究发现BMP7在鸡胚胎的下丘脑大量表达。TAKEDA等[51]研究表明BMPs家族成员在人垂体中有表达。也有研究表明BMPs家族成员在湖羊的子宫[39,48]和卵巢[40]中有表达。VINODKUMAR等[52]研究发现在哺乳动物的输卵管上皮细胞中存在BMPs家族成员的相关受体。上述文献结果表明BMPs家族成员在生物体性腺轴相关组织有一定的表达量。在本试验中与激素分泌相关的下丘脑、垂体、卵巢等组织中均有BMP2,6,7表达。OTSUKA等[53]在小鼠体内研究发现,BMP7能够抑制孕酮含量的上升。绵羊的体外实验研究表明直接卵巢灌注BMP6会导致抑制素A、孕酮和雌激素含量的快速变化以及排卵前LH峰的提前[5]。VINOD等[54]在体外直接培养绵羊颗粒细胞并用不同剂量BMP2处理颗粒细胞,发现不同的剂量均能增加雌激素含量。上述文献表明BMPs家族的成员对动物各种激素或激素受体都有较明显的调控作用。综上,由于BMPs家族基因在分泌性激素的垂体、下丘脑等组织中有表达,且该基因家族对激素具有调控作用,故推测BMP2、BMP6、BMP7对绵羊繁殖力具有一定的调控作用。
3.2 CART和CAST对动物繁殖的调控
研究发现CART在蛋鸡[55]、猪[56]以及小鼠[57]的下丘脑中有表达;在体外培养的小鼠垂体细胞中也发现了CART的表达,同时研究人员也发现该基因的不同剂量可以明显影响PRL/ACTH/TSH和GH的含量[58]。在本试验中CART在小尾寒羊和苏尼特羊的下丘脑和垂体中均有表达,并且该基因在苏尼特羊（单羔）两种组织的表达量均高于小尾寒羊（多羔）。李鹏飞等[58,59]研究发现该基因对FSH诱导的卵泡细胞中的雌激素含量具有一定的抑制作用,由于下丘脑和垂体是性腺轴两个重要的促性腺激素分泌组织,结合本试验结果,进一步为CART抑制雌激素的分泌提供了证据。若在蛋鸡中导入干扰CART表达的dsRNA,发现其对卵泡细胞分泌雌激素具有促进作用[60]。综上推测该基因与雌激素的分泌呈一定的负相关。李鹏飞等[60]研究发现该基因在蛋鸡卵巢中有表达,在本试验中,在两种绵羊的卵巢组织中该基因均呈痕量表达,但JONES等[57]研究发现该基因在小鼠的卵巢内不表达。其可能原因是物种之间的差异,或者所处的生理条件不同。张菊等[61]利用半定量RT-PCR方法研究发现CAST的2型转录本在陶赛特羊卵巢和大脑中有表达。在本实验中CAST在两种绵羊的大脑中均有表达,且该基因在小尾寒羊卵巢中的表达量显著高于苏尼特羊（P＜0.01）,与上述文献结果基本一致。KAPPES等[62,63]研究发现CAST位于牛科动物的7号染色体上,并且在该区域存在潜在的与产双羔相关的基因座。BYUN等[64]通过对大群体统计分析,发现CAST与奶牛繁殖活动以及寿命长短有关;但是GARCIA等[65]研究发现,该基因与罗姆尼羊、美利奴羊、考力代羊的繁殖力以及寿命不存在明显的相关性。在本研究中CAST在小尾寒羊和苏尼特羊子宫、卵巢、输卵管中表达量较高,并且在小尾寒羊子宫和输卵管中的表达量极显著高于苏尼特羊（P＜0.01）,故推测该基因对绵羊的繁殖力具有一定的影响。目前,关于该基因影响动物繁殖力相关的研究几乎处于空白,所以对该基因影响绵羊繁殖力的机制需要进一步探究。
4 结论
本试验利用实时荧光定量PCR探究了BMP2、BMP6、BMP7、CAST和CART在产多羔的小尾寒羊和产单羔的苏尼特羊与性腺轴相关的7种组织中的表达量差异,研究结果表明上述5个基因在单、多羔性腺轴相关组织中的表达有较大差异,暗示这5个基因可能对绵羊繁殖力具有一定的调控作用,为进一步阐明绵羊高繁殖力的分子机理提供了新的思路。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
DOI:10.1530/REP-16-0657URLPMID:28115581 [本文引用: 3]
We hypothesised that cocaine- and amphetamine-regulated transcript (CARTPT) would be differentially expressed in ewes with differing ovulation rates. Expression of mRNA for CARTPT, as well as LHCGR, FSHR, CYP19A1 and CYP17A1 was determined in antral follicles ≥165mm in diameter collected during the follicular phase in ewes heterozygous for the Booroola and Inverdale genes (I+B+; average ovulation rate 4) and ++ contemporaries (++; average ovulation rate 1.8). In ++ ewes (n65=656), CARTPT was expressed in small follicles (1 to <365mm diameter), where 18.865±652.5% follicles expressed CARTPT CART peptide was also detected in follicular fluid of some follicles of ++ ewes. In I+B+ ewes, 5/6 ewes did not have any follicles that expressed CARTPT, and no CART peptide was detected in any follicle examined. Expression pattern of CYP19A1 differed between I+B+ and ++ ewes with an increased percentage of small and medium follicles (3 to <4.565mm diameter) but decreased percentage of large follicles (≥4.565mm diameter) expressing CYP19A1 in the I+B+ ewes. Many of the large follicles from the I+B+ ewes appeared non-functional and expression of LHCGR, FSHR, CYP17A1 and CYP19A1 was less than that observed in ++ ewes. Expression of FSHR and CYP17A1 was not different between groups in small and medium follicles, but LHCGR expression was approximately double in I+B+ ewes compared to that in ++ ewes. Thus, ewes with high ovulation rates had a distinct pattern of expression of CARTPT mRNA and protein compared to ewes with normal ovulation rates, providing evidence for CART being important in the regulation of ovulation rate.
DOI:10.1530/REP-12-0509URLPMID:23801782 [本文引用: 1]
Livestock populations have been subjected to strong selection pressure to improve reproductive success, and this has led to the identification of lines of animals with increased fecundity. These animals provide a rich biological resource for discovery of genes and regulatory mechanisms that underpin improved reproductive success. To date, three genes, all related to the transforming growth factor 13 pathway, have been identified as having mutations that lead to alterations in ovulation in sheep. In addition, several other sheep lines have been identified with putative mutations in single genes with major effects on ovulation rate. This review is focused on the identification of the mutations affecting ovulation rate and how these discoveries have provided new insights into control of ovarian function.
[本文引用: 1]
DOI:10.1095/biolreprod64.4.1225URLPMID:11259271 [本文引用: 2]
The Booroola fecundity gene (FecB) increases ovulation rate and litter size in sheep and is inherited as a single autosomal locus. The effect of FecB is additive for ovulation rate (increasing by about 1.6 corpora lutea per cycle for each copy) and has been mapped to sheep chromosome 6q23-31, which is syntenic to human chromosome 4q21-25. Bone morphogenetic protein IB (BMP-IB) receptor (also known as ALK-6), which binds members of the transforming growth factor-beta (TGF-beta) superfamily, is located in the region containing the FecB locus. Booroola sheep have a mutation (Q249R) in the highly conserved intracellular kinase signaling domain of the BMP-IB receptor. The mutation segregated with the FecB phenotype in the Booroola backcross and half-sib flocks of sheep with no recombinants. The mutation was not found in individuals from a number of sheep breeds not derived from the Booroola strain. BMPR-IB was expressed in the ovary and in situ hybridization revealed its specific location to the oocyte and the granulosa cell. Expression of mRNA encoding the BMP type II receptor was widespread throughout the ovary. The mutation in BMPR-IB found in Booroola sheep is the second reported defect in a gene from the TGF-beta pathway affecting fertility in sheep following the recent discovery of mutations in the growth factor, GDF9b/BMP15.
DOI:10.1095/biolreprod.109.076653URLPMID:19641181 [本文引用: 3]
Bone morphogenetic protein 6 (BMP6) has been suggested as an important local factor capable of modulating the stimulatory actions of follicle-stimulating hormone in granulosa cells in vitro. The objective of this experiment was to determine the effect of direct ovarian infusion of BMP6 (2 渭g/h) on ovarian function in ewes with an autotransplanted ovary. Treated ewes (n = 6) and vehicle-treated controls (n = 6) were infused during the early follicular phase, between 12 and 24 h after luteal regression, and ovarian response was determined by collection of samples of ovarian venous blood and transdermal ultrasound. In the absence of any change in circulating gonadotropins or in the antral follicle population, BMP6 infusion resulted in acute but transient increases in ovarian inhibin A, androstenedione, and estradiol secretion (P < 0.05) during the second half of the infusion period. Thereafter, treated animals had an advance in the time of the LH surge by around 10 h (43.3 卤 2.8 h in treated vs. 53.3 卤 2.7 h...
DOI:10.1530/rep.1.00958URLPMID:16514193
The intraovarian roles of BMP family members such as BMP2, 4, 6 and 7 are not well understood, particularly in species with low ovulation rates such as sheep. Therefore, the objectives of these experiments were to determine the expression patterns of mRNAs encoding BMP2, 4, 6 and 7 during ovarian follicular development in sheep, and to determine the effects of these growth factors on ovine granulosa cell functions in vitro. For comparative purposes, the effects of these BMPs were also determined in rat granulosa cells since these factors have been most widely studied in this poly-ovulatory species. As assessed by in situ hybridization, non-atretic ovine follicles expressed mRNA for BMP6 but not 2, 4 or 7. Furthermore, expression of BMP6 was limited to the oocyte of primordial as well as primary, pre-antral and antral follicles. Reverse transcription-PCR of granulosa cell mRNA detected low levels of all the BMPs in some pools of cells. BMP2, 4, 6 and 7 each inhibited progesterone production from ovine granulosa cells without affecting cellular proliferation/survival. Similarly, these BMPs inhibited progesterone production from rat granulosa cells. However, they also stimulated cellular proliferation/survival of the rat granulosa cells highlighting a species-specific difference for these growth factors. In conclusion, in sheep, BMP2, 4, 6 and 7 inhibit granulosa cell differentiation without affecting proliferation. However, as BMP2, 4 and 7 were not detectable by in situ hybridization in any cells of non-atretic ovarian follicles, it seems unlikely that these proteins would have an important intra-ovarian role in regulating follicular development in sheep. In contrast, localization of BMP6 mRNA in the oocyte suggests that this BMP family member may have a paracrine and/or autocrine role in regulating follicular growth in sheep, as has been shown for two other oocyte derived from members of the transforming growth factor superfamily, BMP15 and growth differentiation factor 9.
Magsci [本文引用: 3]
The genotypes for calpastatin (CAST) and calpain (CAPN) loci were determined by PCR-RFLP and PCR-SSCP methods. Blood samples were collected from 200 pure-bred Zel sheep from Shirang's Zel sheep Breeding Station located in south-west of Golestan, Iran. Extraction of genomic DNA was performed based on the modified salting out method. Based on results, two investigated loci were polymorphic and had different gene variants. Heterozygosis was low for both loci. Chi-square test confirmed Hardy-Weinberg equilibrium only in CAST locus using SSCP method. Detected polymorphisms and associations of genetic variation with meat production and tenderness may help to find the effective genotypes of Zel sheep for the economic traits.
DOI:10.1073/pnas.96.13.7282URLPMID:10377406 [本文引用: 2]
Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor 尾 superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.
DOI:10.1016/S0092-8674(00)81296-9URLPMID:8674122 [本文引用: 1]
Cell. 1996 Jun 28;85(7):947-50. Review
[本文引用: 2]
[本文引用: 1]
DOI:10.1016/j.ydbio.2017.03.002URLPMID:28284906 [本文引用: 1]
Abstract In multicellular organisms, the specification, maintenance, and transmission of the germ cell lineage to subsequent generations are critical processes that ensure species survival. A number of studies suggest that the Bone Morphogenetic Protein (BMP) pathway plays multiple roles in this cell lineage. We wished to use a comparative framework to examine the role of BMP signaling in regulating these processes, to determine if patterns would emerge that might shed light on the evolution of molecular mechanisms that may play germ cell-specific or other reproductive roles across species. To this end, here we review evidence to date from the literature supporting a role for BMP signaling in reproductive processes across Metazoa. We focus on germ line-specific processes, and separately consider somatic reproductive processes. We find that from primordial germ cell (PGC) induction to maintenance of PGC identity and gametogenesis, BMP signaling regulates these processes throughout embryonic development and adult life in multiple deuterostome and protostome clades. In well-studied model organisms, functional genetic evidence suggests that BMP signaling is required in the germ line across all life stages, with the exception of PGC specification in species that do not use inductive signaling to induce germ cell formation. The current evidence is consistent with the hypothesis that BMP signaling is ancestral in bilaterian inductive PGC specification. While BMP4 appears to be the most broadly employed ligand for the reproductive processes considered herein, we also noted evidence for sex-specific usage of different BMP ligands. In gametogenesis, BMP6 and BMP15 seem to have roles restricted to oogenesis, while BMP8 is restricted to spermatogenesis. We hypothesize that a BMP-based mechanism may have been recruited early in metazoan evolution to specify the germ line, and was subsequently co-opted for use in other germ line-specific and somatic reproductive processes. We suggest that if future studies assessing the function of the BMP pathway across extant species were to include a reproductive focus, that we would be likely to find continued evidence in favor of an ancient association between BMP signaling and the reproductive cell lineage in animals. Copyright 2017 Elsevier Inc. All rights reserved.
DOI:10.1016/j.bbamcr.2009.09.012URLPMID:19782107 [本文引用: 1]
GDF5 and BMP2, members of the TGF-尾 superfamily of growth factors, are known to regulate apoptosis in different cell types either positively or negatively. We wanted to investigate the effects of GDF5 and BMP2 on vascular smooth muscle cells and mouse embryonic fibroblasts and disclose the mechanism by which GDF5 and BMP2 might exert anti-apoptotic effects. The effect of GDF5 and BMP2 on proliferation and/or programmed cells death was assessed in isolated human vascular smooth muscle cells and mouse embryonic fibroblasts. We demonstrate that GDF5 and BMP2 prevent apoptosis induced by serum starvation in mouse embryonic fibroblasts but not in smooth muscle cells via the BMP receptor 2 (BMPR2), which is often mutated in hereditary cases of primary pulmonary hypertension. GDF5 and BMP2 stimulate the interaction of BMPR-2 with XIAP thereby reducing the ubiquitination of XIAP, which results in enhanced protein stability. The increased concentration of XIAP counteracts apoptosis by binding and inactivating activated caspases. We conclude that the inhibition of apoptosis in mouse embryonic fibroblasts by BMP2 and GDF5 does not depend on more complex signal transduction pathways such as smad and MAPK signaling but on direct stabilization of XIAP by BMPR2.
DOI:10.1074/jbc.M007428200URLPMID:10998422 [本文引用: 1]
Abstract In developing ovarian follicles, the regulation of cell proliferation and differentiation is tightly coordinated. Precisely how this coordination is achieved is unknown, but recent observations have suggested that molecules emitted by the oocyte are involved in the process. The newly discovered oocyte-specific growth factor, bone morphogenetic protein-15 (BMP-15), is one such molecule. At present, nothing is known about the target cells and biological functions of BMP-15. To fill this gap in our knowledge, recombinant BMP-15 and its antibody were produced and used to determine BMP-15 expression and bioactivity. BMP-15 mRNA and protein were shown to be co-expressed in oocytes throughout folliculogenesis, supporting the idea that BMP-15 is a physiological regulator of follicle cell proliferation and/or differentiation. To test this, we used primary cultures of rat granulosa cells (GCs). We found that BMP-15 is a potent stimulator of GC proliferation, and importantly, the mitogenic effect was follicle-stimulating hormone (FSH)-independent. By contrast, BMP-15 alone had no effect on steroidogenesis. However, it produced a marked decrease in FSH-induced progesterone production, but had no effect on FSH-stimulated estradiol production. This result indicates that BMP-15 is a selective modulator of FSH action. In summary, this study identifies GCs as the first target cells for BMP-15. Moreover, it identifies the stimulation of GC proliferation and the differential regulation of two crucial steroid hormones as the first biological functions of BMP-15. Significantly, BMP-15 is the first growth factor that can coordinate GC proliferation and differentiation in a way that reflects normal physiology.
DOI:10.14670/HH-11-674URLPMID:26435174
This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (P<0.05) increase in follicular diameter after 18 days of culture. However, both FSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5.
DOI:10.1016/j.theriogenology.2017.12.032URLPMID:29331831 [本文引用: 1]
Abstract This study evaluated the effect of bone morphogenetic proteins 2 (BMP2) and 4 (BMP2) on follicle development and mRNA expression for GDF9, Cyclin B1, BMPR1A, BMPR1B, BMPRII, FSHR and SMAD1 in bovine secondary follicles cultured in0002vitro. Isolated secondary follicles were cultured for 18 days in TCM199 + medium alone or supplemented with BMP2 (10090004ng/mL), BMP4 (100090004ng/mL) or combination of both BMP2 and 4. Real-time PCR was used to analyze mRNA levels in fresh and cultured follicles. After 18 days of culture, follicles cultured with BMP2 alone or with BMP4 alone had larger diameters when compared to control (P090004<090004.05). In addition, all treatments promoted antrum formation and maintained a high viability rate through the growing period. The presence of BMP2, BMP4 or both together did not influence mRNA expression for the tested genes. However, the in0002vitro culture induces down-regulation for mRNA expression of BMPR1A. In conclusion, the addition of BMP2 or BMP4 alone in cultured medium promotes follicular growth and antrum formation in bovine follicles after 18 days of in0002vitro culture.
DOI:10.1016/j.theriogenology.2016.12.023URL [本文引用: 1]
61ESR1, ESR2, and PRKAA1 were highly expressed in large follicles of higher-AFC.61Expression of BMP7 and BMPR2 was different between high- and low-AFC ovaries.61Expression of BMP15 was very strongly correlated with BMPR2 in ovary tissues.61Expression of Cyp19 was highly associated with ESR1 in large follicles.61Expression of Cyp19 and PRKAA1 was correlated with BMPs in large follicles.
DOI:10.18632/oncotarget.13259URLPMID:27835610 [本文引用: 1]
The immune response is determined by the speed of the T cell reaction to antigens assured by a state of readiness for proliferation and cytokine secretion. Proliferation, apoptosis and motion of many cell types are controlled by cytoplasmic proteases - μ- and m-calpain - and their inhibitor calpastatin, together forming the “calpain-calpastatin system” (CCS), assumed to modify their targets only upon activation-dependent cytoplasmic Ca2+increase. Contrastingly to this notion, using quantitative real time PCR and semiquantitative flow cytometry respectively, we show here that the CCS genes areconstitutively expressed, and that both calpains areconstitutively activein resting, circulating human CD4+and CD8+lymphocytes. Furthermore, we demonstrate that calpain inhibition in the resting T cells prevents them from proliferationin vitroand greatly reduces secretion of multiple cytokines. The mechanistic reason for these effects of calpain inhibition on T cell functions might be the demonstrated significant reduction of the expression of active (phosphorylated) upstream signalling molecules, including the phospholipase C gamma, p56Lck and NFκB, in the inhibitor-treated cells. Thus, we propose that the constitutive, self-regulatory calpain-calpastatin system activity in resting human T cells is a necessary, controlling element of their readiness for complex and effective response to antigenic challenge.
[本文引用: 1]
DOI:10.1103/PhysRevC.86.014319URL [本文引用: 2]
Calpastatin (CAST) is a specific inhibiter of calpains, playing a role in meat tenderization and myogenesis. In the present study, the polymorphism of the CAST gene of Makoei sheep was investigated by polymerase chain reaction and single strand conformation polymorphism technique (PCR鈥揝SCP). Genomic DNA was extracted from whole blood samples collected from 100 sheep. A 622 bp CAST exon 1 segment was amplified by standard PCR, using the locus specific primers. PCR products were subjected to a non-denaturing gel electrophoresis. Four SSCP patterns, representing four different genotypes, were identified. The frequencies of the observed genotypes were 0.31, 0.04, 0.63 and 0.02 for AA, BB AB and AC, respectively. Allele frequencies were 0.6313, 0.3586 and 0.01 for A, B and C, respectively. The Observed heterozygosity (H) value for CAST gene was 0.4728. The chi-square test showed significant (P< 0.01) deviation from Hardy-Weinberg equilibrium for this locus in Makoei sheep population.
DOI:10.1016/j.theriogenology.2018.02.002URL [本文引用: 1]
Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to equalize CAPN1 haplotypes, CAST , and GHR genetic marker frequencies. The objective was to estimate the pleiotropic effects of these carcass quality oriented markers for body weight, reproduction, and first calf performance traits in 174 replacement beef females which were managed under 2 post-weaning development protocols. Heifers were weighed at 11-, 12-, and 13-mo, at first breeding season pregnancy evaluation, and prior to first calving season. Pubertal status was determined at 11-, 12-, and 13-mo of age. Antral follicles were counted, reproductive tracts were scored, and tract dimensions were measured at 13-mo. Body condition and hip height were scored and measured at pregnancy evaluation and prior to calving season. Heifer pregnancy and weaning rates and ordinal birth date were recorded. Calf body weights at birth and weaning were analyzed. Single df linear contrasts for recessive effects of the GHR heterozygous genotype showed significant decreases of 2.53.6% in 11-, 12-, and 13-mo heifer body weights and heifer weight prior to calving. The additive differences between GHR homozygotes were small and not significant for all body weights measured but a 1k difference in calf birth date was significant. For all 13-mo uterine measurements, scores, and antral follicle counts, only the CAST dominance contrast for medium antral follicle count was significant. The CAPN1 haplotype with a strong additive effect for increased beef tenderness also had a significant additive effect on calving date. Heifers homozygous for the tender haplotype calved 7.9 days later than heifers homozygous for the tough haplotype. Most heifer reproductive traits were not significantly affected by CAST and CAPN1 markers that are widely used to improve beef tenderness by selection and breeders should not be concerned with how these markers affect reproduction and other heifer traits with the possible exception of CAPN1 effects on calving date.
DOI:10.1111/j.1439-0531.2008.01142.xURLPMID:18638104 [本文引用: 1]
The selection of a single ovarian follicle for further differentiation and finally ovulation is a shared phenomenon in monovulatory species from different phylogenetic classes. The commonality of dominant follicle (DF) development leads us to hypothesize that mechanisms for DF selection are conserved. This review highlights similarities and differences in follicular wave growth between cows, mares and women, addresses the commonality of the transient rises in FSH concentrations, and discusses the follicular secretions oestradiol and inhibin with their regulatory roles for FSH. In all three species, rising FSH concentrations induce the emergence of a follicle wave and cohort attrition occurs during declining FSH concentrations, culminating in DF selection. Cohort secretions are initially responsible for declining FSH, which is subsequently suppressed by the selected DF lowering it below the threshold of FSH requirements of all other cohort follicles. The DF acquires relative FSH-independence in order to continue growth and differentiation during low (cow, mare) or further declining FSH concentrations (women), and thus may be the one cohort follicle with the lowest FSH requirement due to enhanced FSH signalling. In all three monovulatory species a transition from FSH- to LH-dependence is postulated as the mechanism for the continued development of the selected DF. In addition, FSH and IGF enhance each other's ability to stimulate follicle cell function and access of IGF-I and -II to the type 1 receptor is regulated by IGF binding proteins that are in turn regulated by specific proteases; all of which have been ascribed a role in DF development. No fundamental differences in DF selection mechanisms have been identified between the different species studied. Thus functional studies of the selection of DFs in cattle and mares are also valuable for identifying genes and pathways regulating DF development in women.
DOI:10.5661/RDR-VI-141URLPMID:17491145 [本文引用: 1]
For a follicle to reach dominance, in mono-ovulatory species such as cattle, requires the integration of a number of processes involving both extra-ovarian signals and intra-follicular paracrine and autocrine regulators. Ovarian transplant studies in both cattle and sheep demonstrated that it takes approximately 4 months for primordial follicles to reach dominance. Gonadotrophins are not a prer...
DOI:10.1016/j.anireprosci.2004.05.017URLPMID:15271471 [本文引用: 1]
Our current perspectives on the relationship between the oocyte and its surrounding somatic cells are changing as we gain a greater understanding of factors regulating folliculogenesis. It is now widely accepted that the oocyte plays a very active role in promoting follicle growth and directing granulosa cell differentiation. The oocyte achieves this, in part, by secreting soluble paracrine growth factors that act on its neighboring granulosa cells, which in turn regulate oocyte development. In preantral follicles, the oocyte directs granulosa cells to regulate oocyte growth, and oocytes may also directly drive follicle growth. In antral follicles, the oocyte governs the behaviour of cells in its immediate vicinity, thereby actively regulating its own microenvironment. As such, the oocyte establishes and maintains the distinct cumulus lineage of granulosa cells. This cell interaction, in general, prevents luteinization of cumulus cells by promoting growth, regulating steroidogenesis and inhibin synthesis, and suppressing luteinizing hormone receptor expression. Conversely, mural granulosa cells in antral follicles, which have no direct physical contact with the oocyte and, presumably, experience a more diffuse concentration of oocyte-secreted factors, proceed to a different phenotype. In the ovulating follicle, oocyte-secreted factors also play vital roles in enabling cumulus cell expansion and regulating extracellular matrix stability, thus facilitating ovulation. The identities of these oocyte-secreted growth factors regulating such key ovarian functions remain unknown, although growth differentiation factor-9 (GDF-9), GDF-9B and/or bone morphogenetic protein-6 (BMP-6) are likely candidate molecules, probably forming complex local interactions with other related members of the transforming growth factor-尾 (TGF-尾) superfamily. Elucidating the nature of oocyte鈥搒omatic cell interactions at the various stages of follicle development will have important implications for our understanding of factors regulating folliculogenesis, ovulation rate and fecundity.
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DOI:10.1210/en.2004-0283URLPMID:15271876 [本文引用: 1]
We recently obtained evidence that cocaine- and amphetamine-regulated transcript (CART), a potent anorectic neuropeptide, is expressed in the bovine ovary. The objectives of this study were to characterize bovine ovarian CART and determine its localization, regulation, and regulatory role during follicular development. CART mRNA was detected in stroma of adult ovaries and in large follicles, but was undetectable in several peripheral tissues, fetal ovaries, and corpora lutea. Within the ovary, CART mRNA and peptide were localized to the granulosal layer of some, but not all, antral follicles, with low, but detectable, expression in oocytes and cumulus cells. CART mRNA was undetectable in granulosal cells of dominant ovulatory follicles collected before and after the preovulatory gonadotropin surge, but was detected in the granulosal layer of adjacent subordinate follicles. In addition, amounts of CART mRNA and follicular fluid concentrations of CART peptide were greater in subordinate follicles vs. dominant follicles of the first follicular wave. Furthermore, CART treatment inhibited basal estradiol production, but not progesterone production, by granulosal cells in a dose-dependent fashion, and the effect was dependent on stage of cell differentiation. We conclude that granulosal cell CART expression is temporally regulated and potentially associated with follicle health status, and CART can inhibit granulosal cell estradiol production. Thus, CART may be a novel local regulator of follicular atresia in the bovine ovary.
DOI:10.1152/physiolgenomics.00123.2004URLPMID:15375196 [本文引用: 1]
Abstract The oocyte is a key regulator of ovarian folliculogenesis and early embryonic development. However, the composition of the oocyte transcriptome and identities and functions of key oocyte-specific genes involved in the above processes are relatively unknown. Using a PCR-based cDNA amplification method (SMART technology), we constructed a bovine oocyte cDNA library. Analysis of 230 expressed sequence tags (ESTs) from this library identified 102 unique sequences. Although some correspond to housekeeping genes (e.g., ribosomal protein L15) and some represent genes previously known to be expressed in oocytes and other tissues, most encode for genes whose expression in mammalian oocytes has not been reported previously (e.g., cocaine- and amphetamine-regulated transcript) or genes of unknown function. Sixteen did not show significant sequence similarity to any entries in the GenBank database and were classified as novel. Using over 2,000 unsequenced, randomly selected cDNA clones from the library, we constructed an oocyte microarray and performed experiments to identify genes preferentially expressed in fetal ovary (an enriched source of oocytes) relative to somatic tissues. Eleven clones were identified by microarray analysis with consistently higher expression in fetal ovaries (collected from animals at days 210-260 of gestation) compared with spleen and liver. DNA sequence analysis of these clones revealed that two correspond to JY-1, a novel bovine oocyte-specific gene. The remaining nine clones represent five identified genes and one additional completely novel gene. Increased abundance of mRNA in fetal ovary for five of the six genes identified was confirmed by real-time PCR. Results demonstrate the potential utility of these unique resources for identification of oocyte-expressed genes potentially important for reproductive function.
DOI:10.1038/nrn2493URLPMID:4418456 [本文引用: 1]
Over the past decade or so, CART (cocaine- and amphetamine-regulated transcript) peptides have emerged as major neurotransmitters and hormones. CART peptides are widely distributed in the CNS and are involved in regulating many processes, including food intake and the maintenance of body weight, reward and endocrine functions. Recent studies have produced a wealth of information about the location, regulation, processing and functions of CART peptides, but additional studies aimed at elucidating the physiological effects of the peptides and at characterizing the CART receptor(s) are needed to take advantage of possible therapeutic applications.
DOI:10.1186/1477-7827-4-22URLPMID:16611367 [本文引用: 1]
pAbstract/p pThe ability of ovarian follicles to produce large amounts of estradiol is a hallmark of follicle health status. Estradiol producing capacity is lost in ovarian follicles before morphological signs of atresia. A prominent wave like pattern of growth of antral follicles is characteristic of monotocous species such as cattle, horses and humans. While our knowledge of the role of pituitary gonadotropins in support of antral follicle growth and development is well established, the intrinsic factors that suppress estradiol production and may help promote atresia during follicular waves are not well understood. Numerous growth factors and cytokines have been reported to suppress granulosa cell estradiol production in vitro, but the association of expression of many such factors in vivo with follicle health status and their physiological significance are not clear. The purpose of this review is to discuss the in vivo and in vitro evidence supporting a local physiological role for cocaine and amphetamine regulated transcript, inhibins and low molecular weight insulin like growth factor binding proteins in negative regulation of granulosa cell estradiol production, with emphasis on evidence from the bovine model system./p
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DOI:10.1210/en.2007-0332URLPMID:17569753 [本文引用: 1]
Abstract Regulation of estradiol production, central to ovarian follicular development and reproductive function, is mediated by a complex interaction of pituitary gonadotropins such as FSH with locally produced regulatory molecules. We previously demonstrated a negative association of expression of cocaine-and amphetamine-regulated transcript (CART) with follicle health status and a novel local negative role for CART in regulation of basal estradiol production by bovine granulosa cells. However, effects of CART on FSH-induced estradiol production and the underlying mechanism(s) mediating the physiological actions of CART on granulosa cells are not known. Objectives of the present study were to determine effects of CART on basal and FSH-induced intracellular cAMP levels, aromatase mRNA, estradiol accumulation, calcium signaling, and the intracellular signaling pathways involved using primary cultures of bovine granulosa cells. CART treatment potently inhibits the FSH-induced rise in granulosa cell cAMP levels, estradiol accumulation, and aromatase mRNA. Furthermore, results show that calcium is essential for FSH-induced cAMP and estradiol accumulation, and CART significantly inhibits FSH-induced calcium influx. Select G protein and protein kinase inhibitors were used to elucidate pathways involved in CART actions. The inhibitory actions of CART on FSH signaling and estradiol production are mediated via a G(o/i)-dependent pathway, whereas none of the other signaling inhibitors had any effect on CART actions. Results demonstrate novel potent inhibitory effects of CART on multiple components of the FSH signaling pathway linked to estradiol production and follicular development and shed new insight into the mechanism of action of CART potentially pertinent within and beyond the reproductive system.
DOI:10.1095/biolreprod.109.077586URLPMID:19439726 [本文引用: 1]
We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1 mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPT mRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1 mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPT mRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPT mRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1 mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.
DOI:10.1210/en.2004-0283URLPMID:15271876 [本文引用: 1]
We recently obtained evidence that cocaine- and amphetamine-regulated transcript (CART), a potent anorectic neuropeptide, is expressed in the bovine ovary. The objectives of this study were to characterize bovine ovarian CART and determine its localization, regulation, and regulatory role during follicular development. CART mRNA was detected in stroma of adult ovaries and in large follicles, but was undetectable in several peripheral tissues, fetal ovaries, and corpora lutea. Within the ovary, CART mRNA and peptide were localized to the granulosal layer of some, but not all, antral follicles, with low, but detectable, expression in oocytes and cumulus cells. CART mRNA was undetectable in granulosal cells of dominant ovulatory follicles collected before and after the preovulatory gonadotropin surge, but was detected in the granulosal layer of adjacent subordinate follicles. In addition, amounts of CART mRNA and follicular fluid concentrations of CART peptide were greater in subordinate follicles vs. dominant follicles of the first follicular wave. Furthermore, CART treatment inhibited basal estradiol production, but not progesterone production, by granulosal cells in a dose-dependent fashion, and the effect was dependent on stage of cell differentiation. We conclude that granulosal cell CART expression is temporally regulated and potentially associated with follicle health status, and CART can inhibit granulosal cell estradiol production. Thus, CART may be a novel local regulator of follicular atresia in the bovine ovary.
DOI:10.1016/S1357-2725(98)00110-1URLPMID:9924797 [本文引用: 1]
Abstract Cocaine and amphetamine regulated transcript peptide (CART), is a recently discovered hypothalamic peptide with a potent appetite suppressing activity. In the rat the CART gene encodes a peptide of either 129 or 116 amino acid residues whereas only the short form exists in humans. The predicted signal sequence is 27 amino acid residues resulting in a prohormone of 102 or 89 residues. The C-terminal end of CART, consisting of 48 amino acid residues and 3 disulphide bonds, is thought to constitute a biologically active part of the molecule. In the central nervous system CART is highly expressed in many hypothalamic nuclei, some of which are involved in regulating feeding behaviour. The CART mRNA is regulated by leptin, and the expressed CART is a potent inhibitor of feeding that even overrides the feeding response induced by neuropeptide Y. The putative CART receptor is therefore a potential therapeutic target for an anti-obesity drug.
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DOI:10.1124/jpet.105.091512URLPMID:16840648 [本文引用: 1]
Abstract CART (cocaine- and amphetamine-regulated transcript) peptides are neuromodulators that are involved in feeding, drug reward, stress, cardiovascular function, and bone remodeling. CART peptides are abundant but discretely distributed in the brain, pituitary and adrenal glands, pancreas, and gut. High expression of CART in discrete hypothalamic nuclei associated with feeding has led to behavioral and pharmacological studies that strongly support an anorectic action of CART in feeding. Subsequent studies on humans and transgenic animals provide additional evidence that CART is important in the regulation of appetite as mutations in the CART gene are linked to eating disorders, including obesity and anorexia. The expression of CART in the mesolimbic dopamine circuit has lead to functional studies demonstrating CART's psychostimulant-like effects on locomotor activity and conditioned place preference in rats. These and other findings demonstrated that CART modulates mesolimbic dopamine systems and affects psychostimulant-induced reward and reinforcing behaviors. The link between CART and psychostimulants was substantiated by demonstrating alterations of the CART system in human cocaine addicts. CART seems to regulate the mesolimbic dopamine system, which serves as a common mechanism of action for both feeding and addiction. Indeed, recent studies that demonstrated CART projections from specific hypothalamic areas associated with feeding to specific mesolimbic areas linked to reward/motivation behaviors provide evidence that CART may be an important connection between food- and drug-related rewards. Given the enormous public health burden of both obesity and drug addiction, future studies exploring the pharmacotherapies targeting CART peptide represent an exciting and challenging research area.
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DOI:10.1210/mend.12.12.0206URLPMID:9849956 [本文引用: 1]
We have taken advantage of the sequence relationships among the bone morphogenetic proteins (BMPs) to identify the mouse Bmp15 and human BMP15 genes. The 392-amino acid prepropeptides encoded by these BMP genes exhibit significant homology to each other, although the 70% identity observed between the 125-amino acid mature peptides is considerably lower than that seen in comparisons of other mouse and human orthologs. Both genes share a common structural organization and encode mature peptides that lack the cysteine residue normally involved in the formation of a covalent dimer. In addition, mouse Bmp15 and human BMP15 map to conserved syntenic regions on the X chromosome. We demonstrate, through a combination of Northern blot and in situ hybridization analyses, that mouse Bmp15 is expressed specifically in the oocyte beginning at the one-layer primary follicle stage and continuing through ovulation. Interestingly, BMP-15 is most closely related to and shares a coincident expression pattern with the mouse growth/differentiation factor 9 (GDF-9) gene that is essential for female fertility. Our findings will be important for defining the role of BMP-15 in follicular development.
DOI:10.1006/bbrc.1996.0889URLPMID:8670277
BMP-3b (also termed GDF-10) is a novel BMP-3 related protein recently discovered in rat femur tissue by molecular cloning. In this paper, we have isolated cDNA and the gene for human BMP-3b and determined their structure. Cloned human BMP-3b cDNA with a size of 2632 base pairs encodes a 478 amino acid precursor protein sharing 83% identity with rat BMP-3b. The human BMP-3b gene is composed of 3 exons and spans approximately 13 kilobases of DNA. The 5-flanking region carries no typical TATA box but G+C rich sequences. Southern blot analysis revealed that the BMP-3b gene is situated in a single locus of chromosome 10. By Northern analysis, human BMP-3b transcripts were detected primarily in femur, brain, lung, skeletal muscle, pancreas and testis.
DOI:10.1210/jc.81.11.3877URLPMID:8923832
Bone () belong structurally to the transforming -beta superfamily comprising several and differentiation factors such as inhibin, activin, and Mllerian inhibitory factor that regulate ovarian function. We studied here the potential expression of , -3, and -4 messenger RNAs (mRNAs) in isolated granulosa obtained at oocyte retrieval for in vitro . Freshly isolated granulosa were found to express (also known as ) mRNAs but not those of or -4. The transcripts were detected with RT-PCR amplification followed by Southern blot hybridization, as well as by Northern and dot blot hybridization analyses. To investigate whether mRNAs are hormonally regulated, cultures of granulosa-luteal (GL) were treated with different concentrations of purified (hCG) at varying stages of culture. hCG decreased mRNA levels from the first day of the culture up to day 5. Time-dependence studies showed that a clear decrease in mRNA levels was evident at 24 h after hCG treatment, and that the effect of hCG was concentration dependent with 3 ng/mL hCG decreasing significantly (P < 0.05) mRNA levels. Furthermore, the cAMP analog, 8--cAMP (8-Br-cAMP), which activates , and 12-0-tetradecanoylphorbol 13-, an activator of , both markedly decreased mRNA levels in an 8-h treatment. We conclude that: 1) mRNAs are expressed in granulosa ; 2) their steady state levels are hormonally regulated in cultured GL as evidenced by the ability of hCG to markedly decrease transcript levels; and (3) that activation of both -and -mediated pathways also results in a decrease in mRNA levels in GL . We suggest that , like several other members of the transforming -beta superfamily, is a potential local regulator of female gonadal function.
DOI:10.1101/gad.3.11.1657URLPMID:2481605
The -1 (Vg-related) and -2a (bone 2a) genes are members of the decapentaplegic subgroup of the transforming growth factor-beta (TGF beta) superfamily. Although genetic and biochemical studies suggest that the members of this subgroup play important roles in , little is known about their function in . Therefore, we investigated the expression of -1 and -2a in , newborn, and adult tissues by in situ hybridization. -1 RNA is maternally encoded in ovarian oocytes but declines in fertilized eggs and is undectable by the two- to four-cell stage. Only low levels of transcripts are seen in blastocysts and early postimplantation stages. From mid-on, -1 RNA is expressed at high levels in developing skin, especially in the suprabasal cells of the proliferating epidermis but not in the dermis or hair follicles, both of which contain and/or . In contrast, -2a transcripts are seen only in the hair follicles in the cells of the hair bulb cortex. Temporally and spatially distinct patterns of -2a, -1, , and expression are also seen in different populations of mesenchymal cells in the developing skeletal system (cartilage and bone). Our results suggest that the coordinated expression of several members of the TGF beta superfamily is required to control the progression of specific cell types through their differentiation pathways.
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DOI:10.1530/rep.0.1230363URLPMID:11882013 [本文引用: 1]
The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.
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DOI:10.1530/REP-15-0315URLPMID:27486268 [本文引用: 1]
Abstract Cystic ovarian disease (COD) is an important cause of subfertility in dairy cattle. Bone morphogenetic proteins (BMPs), mainly BMP2, BMP4 and BMP6, play a key role in female fertility. In this study, we hypothesized that an altered BMP system is associated with ovarian alterations contributing to COD pathogenesis. Therefore, we examined the expression of BMP2, BMP4 and BMP6 and BMP receptor 1B (BMPR1B) in the ovaries of animals with spontaneous or ACTH-induced COD, as well as during the development of the disease, in a model of follicular persistence induced by low doses of progesterone (at 5, 10 and 15 days of follicular persistence). Results showed changes in BMP2, BMP4 and BMP6 expression during folliculogenesis, in granulosa and theca cells in the COD groups, as well as at different stages of follicular persistence. Results also showed changes in BMPR1B expression in developing follicles in animals with COD, and at the initial stages of follicular persistence (P5). Comparison between groups showed significant differences, mainly in BMP4 and BMP6 expression, in granulosa and theca cells of different follicular categories. The expression of these BMPs also increased in cystic and persistent follicles, in relation to antral follicles of the control group. BMPR1B showed high expression in cystic follicles. Together, these results may indicate an alteration in BMPs, especially in BMP4 and BMP6, as well as in BMPR1B, which occurs early in folliculogenesis and incipiently during the development of COD, which could be a major cause of recurrence of this disease in cattle.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/early/2016/08/01/REP-15-0315/suppl/DC1. 2016 Society for Reproduction and Fertility.
DOI:10.1016/j.neulet.2013.08.027URLPMID:23978511 [本文引用: 1]
Hypothalamus plays a key role in homeostasis, and functions of the hypothalamus depend on the accurate trajectory of hypothalamic neuroendocrine axons. Thus, understanding the guidance of hypothalamic neuroendocrine axons is crucial for knowing how hypothalamus works. Previous studies suggest FGF10 deriving from the medial ventral midline of the hypothalamus plays an important role in axon guidance of the developing hypothalamus. Here we show that Shh and BMP7, which are from the anterior and posterior hypothalamic ventral midline respectively, together repel hypothalamic axons towards the medial ventral midline.
DOI:10.1016/S0006-291X(03)01052-0URLPMID:12821114 [本文引用: 1]
Roles of activin/bone morphogenetic protein (BMP) system in the pathogenesis of human pituitary adenoma remain unknown although these factors stimulate follicle-stimulating hormone (FSH) secretion in the normal pituitary. Here we demonstrated that type-I and -II subunit mRNAs of activin/BMP receptors are expressed in Pit-1-negative FSH-producing (FSH-oma) and nonfunctioning pituitary adenomas (NF-oma). Basal levels of serum FSH standardized by luteinizing hormone (LH) were markedly high in FSH-omas in contrast to NF-omas. However, gonadotropin-releasing hormone (GnRH)-induced increment of FSH standardized by that of LH was not changed in FSH-omas, suggesting that imbalanced FSH secretion by FSH-oma is not attributable to GnRH regardless of the expression of GnRH receptor. Although activin 尾A subunit was detected in neither adenoma, the 尾B subunit was expressed highly in FSH-omas and, to lesser extent, in NF-omas. As for BMPs, BMP-6 and -7 were detected in NF-omas while BMP-4 and -15 were not detected in either type of adenoma. In the presence of pituitary activin/BMP system, the levels of co-expressing follistatin mRNA in the tumors were reduced in FSH-oma compared with NF-oma, suggesting that endogenous follistatin is involved in FSH overproduction through inhibition of activin/BMP system independently of GnRH.
DOI:10.1016/j.anireprosci.2017.08.006URLPMID:28830629 [本文引用: 1]
Members of the transforming growth factor beta (TGF-尾) family, including bone morphogenetic proteins (BMPs), are expressed in the epithelial cells of the mammalian oviduct. These signaling molecules play important roles in development and tissue homeostasis; however, little is known about their function in the mammalian oviduct. In the present study, RT-qPCR was used to analyze the mRNA abundance of BMP type I (BMPR1A, BMPR1B, ACVR1) and type II receptors (BMPR2, ACVR2A, ACVR2B) in the bovine oviduct epithelial cells (BOEC) isolated from ampulla and isthmus at both the follicular (FP) and the luteal (LP) phase of the estrous cycle. Results indicate that mRNAs for all the BMP receptors studied are expressed in the BOEC. Significant mRNA abundance differences were observed for both BMPR1B and ACVR2B when comparing both the ampulla and isthmus regions with the greater abundance at the isthmus. When both FP and LP samples were compared, ACVR2B mRNA showed greater abundance during the LP, with significant differences in the isthmus region. These variations highlight differences between the isthmus and ampulla regions of the oviduct. By means of wound healing assays on BOEC primary cultures, exogenous recombinant human BMP5 induced a significant increase in wound healing at 24h. The observed changes at the mRNA abundance of components of the signaling pathway and the BMP5 effect on oviductal epithelial cells suggest a possible autocrine role for the BMP pathway that could affect epithelial cell functions necessary for normal physiology and reproductive success in BOEC homeostasis.
DOI:10.1074/jbc.M103212200URLPMID:11447221 [本文引用: 1]
Abstract The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. Growth factors emitted by oocytes integrate and promote this process. Growth differentiation factor-9 (GDF-9), bone morphogenetic protein (BMP)-15, and BMP-6 are oocyte-derived members of the transforming growth factor-beta superfamily. In contrast to the recent studies on GDF-9 and BMP-15, nothing is known about the biological function of BMP-6 in the ovary. Here we show that, unlike BMP-15 and GDF-9, BMP-6 lacks mitogenic activity on rat granulosa cells (GCs) and produces a marked decrease in follicle-stimulating hormone (FSH)-induced progesterone (P(4)) but not estradiol (E(2)) production, demonstrating not only the first identification of GCs as BMP-6 targets in the ovary but also its selective modulation of FSH action in steroidogenesis. This BMP-6 activity resembles BMP-15 but differs from GDF-9 activities. BMP-6 also exhibited similar action to BMP-15 by attenuating the steady state mRNA levels of FSH-induced steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage enzyme (P450scc), without affecting P450 aromatase mRNA level, supporting its differential function on FSH-regulated P(4) and E(2) production. However, unlike BMP-15, BMP-6 inhibited forskolin- but not 8-bromo-cAMP-induced P(4) production and StAR and P450scc mRNA expression. BMP-6 also decreased FSH- and forskolin-stimulated cAMP production, suggesting that the underlying mechanism by which BMP-6 inhibits FSH action most likely involves the down-regulation of adenylate cyclase activity. This is clearly distinct from the mechanism of BMP-15 action, which causes the suppression of basal FSH receptor (FSH-R) expression, without affecting adenylate cyclase activity. As assumed, BMP-6 did not alter basal FSH-R mRNA levels, whereas it inhibited FSH- and forskolin- but not 8-bromo-cAMP-induced FSH-R mRNA accumulation. These studies provide the first insight into the biological function of BMP-6 in the ovary and demonstrate its unique mechanism of regulating FSH action.
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DOI:10.1124/jpet.105.096123URLPMID:16322355 [本文引用: 2]
Cocaine-amphetamine-regulated transcript (CART), a neuropeptide involved in the brain's reward/reinforcement circuit, modulates the effects of psychostimulants, including cocaine. The CART gene has been characterized, and binding sites for multiple transcription factors have been identified within the promoter region, including the cAMP-response element, which serves as a binding site for cAMP-response element-binding protein (CREB). CART expression appears to be regulated via cAMP/protein kinase A (PKA)/CREB-mediated signaling in cell culture. Therefore, the goal of these studies was to examine the involvement of cAMP/PKA/CREB-mediated signaling in CART mRNA and peptide expression in vivo in the rat nucleus accumbens. Intra-accumbal injections of forskolin, an adenylyl cyclase activator, stimulated the phosphorylation of CREB and increased both CART mRNA and peptide levels, an effect attenuated by inhibition of PKA with H89 [N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline-sulfonamide hydrochloride] and adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In addition, Rp-cAMPS alone decreased CART mRNA compared with saline-injected controls, suggesting that CART expression may be tonically regulated by PKA. Under certain conditions, cocaine increases CART mRNA levels; thus, we examined the effects of cocaine on forskolin-induced CART mRNA expression in the rat nucleus accumbens. Cocaine plus forskolin significantly increased CART mRNA over either of the drugs administered independently, suggesting that under conditions of heightened cAMP signaling, cocaine may impact CART gene expression. These results suggest that CART expression in vivo in the rat nucleus accumbens is regulated by adenylyl cyclase and cAMP/PKA-mediating signaling and, likely, through the activation of CREB.
DOI:10.1016/j.peptides.2017.03.003URLPMID:28300671 [本文引用: 2]
Objectives The aim of this study is to present the results of transanal one-stage pull-through for Hirschsprung's disease.Methods Fourteen children aging from 22 days to 3 months who underwent transanal one-stage pull-through were reviewed.The technique include a partial rectal mucosectomy at 0.5cm proximal to the dentate line posteriorly and 2cm anteriorly.Resections of the rectal muscularis below the peritoneal reflection was carried out to the level of musculus levator left a short muscular cuff.An oblique anastomosis was made between the pull-through colon and anus.Results The operative duration was 90-150 minutes and an average volume of bleeding was about 25ml.There were no intraoperative or postoperative complications.The patients tolerated feeding on the second postoperative day.All patients had 1-3 bowel movements per day during a period of 1-6 months follow-up. Conclusions This procedure is an easy adaptation to a well-described technique in infants.The long-term results should be followed-up.
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DOI:10.2527/2000.78123053xURLPMID:11132819 [本文引用: 1]
Abstract Genomic scans were conducted with 273 markers on 181 sires from a cattle population selected for increased twinning rate to identify chromosomal regions containing genes that influence ovulation rate. Criteria used for selecting markers were number of alleles, ease of scoring, and relative position within linkage group. Markers were multiplexed or multiple-loaded on the gels to reduce the costs and labor required to obtain genotypic data. This approach reduced the number of gels by 45% when compared with running each marker independently. Male animals selected for the genomic scan sired the majority of the population. A modified interval analysis was used in a granddaughter design to compare effects of each allele within sire for 10 different sire families. The midparent deviation of the son's estimated breeding value for ovulation rate was used as the phenotype. Forty-one potential peaks were identified with a nominal significance level < or = 0.05. The 10 peaks with the highest significance levels (P < 0.02) were selected for further analysis. Markers were genotyped across daughters of the sire where nominal significance was found for each of the 10 peaks. One peak (BTA5, relative position 40 cM) was found to be nominally significant in the daughters. The nominal significance levels were P = 0.01 for the sons (n = 32) and P = 0.02 for the daughters (n = 94) of sire 784403. A combined genomewide significance value (P = 0.07) was calculated that accounted for the 10 analyses with sons and the 10 analyses with daughters. These results strongly suggest that this region contains a gene(s) that is involved in the follicular recruitment and development process.
DOI:10.1007/s003350010180URLPMID:11003703Magsci [本文引用: 1]
An autosomal genome scan for quantitative trait loci (QTL) affecting twinning rate was carried out in the Norwegian Cattle population. Suggestive QTL were detected on Chromosomes (Chr) 5, 7, 12, and 23. Among these, the QTL positions on both Chr 5 and Chr 23 are strongly supported by literature in the field. Our results also confirm previous mapping of a QTL for twinning to Chr 7, but definitely suggest a different location of the QTL on this chromosome. The most convincing QTL peak was observed for a region in the middle part of Chr 5 close to the insulin-like growth factor 1 (IGF1) gene. Since IGF1 plays an important role in the regulation of folliculogenesis, a mutation search was performed by sequencing more than 3.5 kb of the gene in actual families. The sequencing revealed three polymorphisms in noncoding regions of the gene that will be important in fine structure mapping and characterization of the QTL.
DOI:10.1111/j.1365-2052.2006.01443.xURLPMID:16734705 [本文引用: 1]
Anim Genet. 2006 Jun;37(3):304-5. Research Support, Non-U.S. Gov't
DOI:10.1111/j.1365-2052.2009.01982.xURLPMID:19917051 [本文引用: 1]
Department of Agricultural Sciences, Lincoln University, Canterbury, New Zealand.
