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Texel与乌珠穆沁绵羊妊娠中、后期胎儿骨骼肌基因表达谱及组织学分析
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摘要
绵羊是早熟性物种,但胎儿肌纤维增殖主要发生在妊娠的中后期。不同品种绵羊肌肉生长潜力的差异主要取决于其胎儿期的肌纤维增殖情况。本研究以两个骨骼肌表型差异明显的Texel和乌珠穆沁绵羊为实验对象,在它们妊娠的70,85,100,120和135日龄,应用专门化的绵羊oligo DNA微阵列芯片,对其胎儿的背最长肌基因表达谱进行分析,从转录组水平揭示两种不同表型绵羊的内在差异。同时,应用组织学方法对每个品种羊11个骨骼肌组织的生长发育和肌纤维表型进行了分析。主要实验结果如下:
     1. Texel与乌珠穆沁绵羊胎儿体重与骨骼肌发育学变化
     在胎儿70至100日龄阶段,Texel羊体重极显著大于乌珠穆沁胎羊(P<0.01);120和135日龄阶段品种间差异不显著(P>0.05)。两品种绵羊的各个骨骼肌组织生长发育差异具有时段特征。85日龄,半膜肌,背最长肌,股四头肌,臀中肌与趾浅屈肌重量,Texel显著或极显著(P<0.05或P<0.01)大于乌珠穆沁;至100日龄阶段, 12个骨骼肌组织生长发育的差异达到最大(P<0.05或P<0.01),Texel羊占有绝对优势;发育至120和135日龄阶段,仅冈下肌的生长发育在Texel还保持优势(P<0.05);其余11个骨骼肌生长发育在品种间差异不显著(P>0.05)。85与100日龄阶段骨骼肌发育上的这种优势特征为Texel出生后高的肌肉生长发育潜力奠定了物质基础。
     两品种羊胎儿骨骼肌生长发育模式存在一定的差异。肌纤维横截面积发育性变化趋势相似;肌纤维数和密度在不同部位的肌肉有着不同的特征性生长曲线。按骨骼肌纤维的生长发育模式主要分为四类:①背最长肌,腓肠肌,半腱肌,臀中肌和臂三头肌在两品种都有明显的肌纤维增殖,但品种间的增殖时间点不同。②半膜肌,股四头肌和臀股二头肌,在Texel羊有明显的新肌纤维生成,而乌珠穆沁羊未观察到。③内收肌和冈下肌在乌珠穆沁羊有明显的新肌纤维生成,而Texel羊未观察到。④在乌珠穆沁与Texel羊均未观察到冈上肌有明显的新肌纤维生成。
     2. Texel与乌珠穆沁绵羊妊娠中、后期胎儿背最长肌基因表达谱分析
     (1)品种内整个发育阶段基因表达变化。在骨骼肌发育过程中,Texel与乌珠穆沁羊品种内差异基因的功能分类基本相似,主要包括以下几个生物学过程:代谢,转运,细胞通信,外部刺激反应,细胞死亡,形态发生,转录调节,细胞增殖,细胞过程的调节和细胞分化。差异基因所参与的生物学过程存在品种特异性:Texel特异性地富集与DNA,RNA和基因表达调控以及细胞增殖相关的基因;而乌珠穆沁则特异性地富集与脂肪合成代谢,肌肉发育,免疫反应,形态学发生和细胞分化相关的基因。这些品种内特异表达的差异基因可优先作为myostatin的候选靶基因来研究。
     基因表达的动态模式分析发现,两品种绵羊胎儿骨骼肌差异基因具有不同的基因表达谱。Texel的基因表达模式有3个趋势类别,且上调表达的基因数量相对较多;而乌珠穆沁的基因表达模式有4个趋势类别,只有一种上调表达模式。基因网络分析表明,两品种羊骨胳肌发育的基因网络存在差异。Texel羊肌发育的基因网络主要包含“RNA转录后修饰”,“细胞发育”及“骨骼与肌肉系统发育和功能”三个分子功能。而乌珠穆沁羊生肌网络则由“脂代谢”,“小分子生化反应”及“骨骼与肌肉系统发育和功能”三个分子事件构成。
     (2)品种间同一发育阶段基因表达。在每一特定的发育阶段两品种绵羊差异表达基因的功能分类存在差异。各个阶段的差异基因分别显著富集于不同的信号通路和基因网络,也预示着这些分子通路和基因网络与myostatin突变紧密相关,信号通路和基因网络中的差异基因可能是myostatin的靶基因。
     本研究表明,转录组水平上的差异是引起Texel与乌珠穆沁绵羊骨骼肌表型差异的主要原因。
Sheep are large precocial species, and the maximum myofibre complement of a muscle is achieved prior to birth,especially in the second half of gestation in the case of sheep. The potential of growth in muscle depends on the muscle fibre number formed during the prenatal stage. In the present study, first, we examined the patterns of proliferation of myofibre in these two sheep breeds during the second half of gestation .Then, using the commercial and standardized sheep transcriptome-wide oligo DNA microarray (Agilent), we first analyzed the transcriptomic profiles of longissmuss dorsi muscle from fetuses of Texel and Ujumqin sheep, sampled at d70, d85, d100, d120 and d135 of gestation respectively. The main findings are followings:
     1. Dynamic changes of body weight and individual muscle weight during the fetal development in Texel and Ujumqin sheep.
     Weights of fetus and the individual muscle tissue in Texel sheep were significantly higher than Ujumqin sheep at the stage of 70,85 and 100d (P<0.05 or P<0.01), whereas there were no differences between them at the stage of 120 and 135d (P>0.05). There was specific profile of each muscle tissue between two sheep breeds during the skeletal muscle development. Weights of the semimembranosus, longissimus dorsi, quadriceps femoris, gluteus medius and flexor digitorum superficialis muscle in Texel sheep were significantly higher than Ujumqin sheep at 85 day of gestation. Until the stage of 100d, the Texel sheep had the preponderance over the Ujumqin sheep and the differences between them became ever greater than else. When developing at 120 and 135 day of gestation, there were no differences in the remaining 11 muscle tissues except that the infraspinatus in Texel sheep grew faster than Ujumqin sheep (P<0.05). The preponderance of development and growth in Texel at 85 and 100 day of gestation underlied their fast postnatal growth of muscle.
     There were distinct patterns of hyperplasia of muscle fibre between Texel and Ujumqin sheep at the histological level during the second half of gestation. First, there were obvious hyperplasia in musculus longissimus dorsi, gastrocnemius, semitendinosus, gluteus medius and triceps brachii in these two sheep breeds. However the timing of proliferation in myofibre was different between Texel and Ujumqin sheep. Second, there were hyperplasia in semimembranosus, quadriceps femoris and biceps femoris in Texel sheep, but not observed in Ujumqin sheep. Third, there were proliferation of myofibre in adductor and infraspinatus in Ujumqin sheep, but not observed in Texel sheep. Finally, there was no obvious hyperplasia of myofibre observed in supraspinatus in both sheep breeds during the second half of gestation. However, the two sheep breeds had the same patterns of myofibre cross-section area during the skeletal muscle development.
     2. Profiles of gene expression in sheletal muscle between Texel and Ujumqin sheep during the second half of gestation.
     2.1 GO analysis showed that in general, the categories of biological processes involved in myogenesis were similar in Texel and Ujumqin sheep. Mainly, they included the following biological processes: metabolism, transport, cell communication, response to external stimulus, cell death, morphogenesis, regulation of transcription , regulation of cellular process, cell proliferation, cell differentiation. However, Texel and Ujumqin sheep quite differred in certain biological processes for differential genes across the development times. For example, genes related to DNA, RNA , regulation of gene expression and cell proliferation were enriched significantly in the Texel gene set, while genes involved in fat anabolic metabolism, muscle development, immune response, morphogenesis and cell differentiation were enriched significantly in Ujumqin gene set.
     We demonstrated that there were varietal specific gene expression profiles derived from differentially expressed genes in each sheep breed across the five developmental stages. There are 3 and 4 kind of profiles in Texel and Ujumqin sheep, respectively.Also there were more uptrend genes and profiles in Texel, compared with Ujumqin sheep. It suggested that the additional up-regulated genes might be part of target genes of myostatin. We identified two different gene network associated with myogenesis in Texel and Ujumqin sheep across the development stages.Network in Texel sheep is involved in function of“RNA Post-Transcriptional Modification”,“Cellular Development”and“Skeletal and Muscular System Development and Function”, while the one in Ujumqin sheep focuses on“Lipid Metabolism”,“Small Molecule Biochemistry”and“Skeletal and Muscular System Development and Function”.
     2.2 At the each developmental stage between the two breed, comparisons showed that differentially expressed genes were enriched in various signaling pathways or networks, genes in which probably are targets of myostatin in skeletal muscle development, suggesting that perturbation of myostatin can affect several biological processes during the myogenesis. The cellular functions of the differentially expressed probes that matched annotated genes revealed characteristic differences in various categories between two breeds at each developmental stages. The diversity of transcription profiles in prenatal skeletal muscle underlies the phenotype variation in these two sheep groups.
     This present study revealed that there were intrinsic differences in skeletal muscle development on histological level as well as on transcriptomic level between Texel and Ujumqin sheep. Next works are to elucidate the putative networks involved in skeletal muscle development on protein level and to determine direct or indirect target genes of myostatin in the context of development.
引文
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