Abstract:

To investigate the biological functions of SMAD2， SMAD3 and SMAD4 genes in the reproductive activity of small-tailed cold sheep， which are important members of the SMAD gene family， the bioinformatics techniques were used to analyze the physicochemical properties， hydrophilicity and subcellular localisation of the SMAD2， SMAD3 and SMAD4 genes， as well as secondary structures， protein interaction prediction and enrichment analysis of gene ontology （GO） and kyoto encyclopedia of genes and genomes （KEGG）. The small-tailed sheep was as material， positive expression of SMAD2， SMAD3 and SMAD4 proteins in ovarian tissue was determined by immunohistochemistry staining （IHC）， and the real-time quantitative PCR （RT-qPCR） and western blot （WB） were used to determine the expression levels of SMAD2， SMAD3 and SMAD4 mRNAs and their encoded proteins in ovarian tissues and follicles of different diameters at different physiological stages. The results showed that SMAD2， SMAD3 and SMAD4 proteins were hydrophilic proteins with theoretical isoelectric points of 6.67， 6.73 and 6.50. The positive expressions in ovarian tissues at different physiological stages were mainly located in oocytes， follicular membrane cells and granulosa cells， and the highest proportion of α-helices and random nematic clusters in the secondary structure， which were associated with FOXH1， ACVR1B， SMAD1， TGFβR1， TGFβR2， ZFYVE9， SKI， SKIL， TGIF1 and ENSOARP0000001869 proteins. SMAD2， SMAD3 and SMAD4 genes played biological functions in the TGF-β receptor signalling pathway， TGF-β receptor binding， TGF-β activated receptor activity and were enriched in TGF-β signalling pathway. The expressions of SMAD2 and SMAD4 proteins were significantly higher in ovarian tissues at 42 h after bolus withdrawal than those at 18 h after bolus withdrawal， and their mRNA expression showed the consistent trends with proteins. However， the expressions of SMAD3 gene mRNA and its encoded protein were not significant in ovarian tissues between different physiological stages. The expressions of SMAD3 and its encoded protein were significantly higher in medium follicles than those in large follicles， while the expressions of SMAD2 and SMAD4 were no significant differences between different follicle diameters. In conclusion， SMAD2， SMAD3 and SMAD4 genes had different biological functions in ovarian cycling of small-tailed cold sheep， i.e. SMAD2 and SMAD4 genes were involved in ovarian physiological changes from early oestrus to preovulation in small-tailed cold sheep， but not in follicle development； SMAD3 was involved in follicle development. Above results provided a theoretical reference to further explore the molecular mechanism of the SMAD gene family in ovarian activity in small-tailed cold sheep.

Key words: small tail cold sheep, ovary, follicles, SMAD2, SMAD3, SMAD4

摘要：

为探究SMAD基因家族重要成员SMAD2、SMAD3和SMAD4基因在小尾寒羊繁殖活动中的生物学功能，利用生物信息学技术对SMAD2、SMAD3和SMAD4 基因的蛋白理化性质、亲疏水性和亚细胞定位进行分析，并对其二级结构、蛋白互作进行预测及GO（gene ontology）和KEGG（kyoto encyclopedia of genes and genomes）的富集分析；然后以小尾寒羊为研究对象，通过免疫组织化学染色（immunohistochemistry，IHC）确定SMAD2、SMAD3和SMAD4蛋白在卵巢组织中的表达，利用实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）和蛋白免疫印迹（western blot，WB）方法测定SMAD2、SMAD3和SMAD4基因mRNA及其编码蛋白在不同生理阶段卵巢组织和不同直径卵泡中表达水平。结果显示，SMAD2、SMAD3和SMAD4蛋白为亲水性蛋白，理论等电点分别为6.67、6.73和6.50，在不同生理阶段卵巢组织中的阳性表达主要位于卵母细胞、卵泡膜细胞和颗粒细胞，且二级结构中α螺旋和无规则卷曲的占比较高，其蛋白与FOXH1、ACVR1B、SMAD1、TGFβR1、TGFβR2、ZFYVE9、SKI、SKIL、TGIF1和ENSOARP0000001869蛋白存在互作关系。SMAD2、SMAD3和SMAD4基因在TGF-β受体信号通路、TGF-β受体结合、TGF-β激活受体活性中发挥生物学功能，且在TGF-β信号通路富集。SMAD2和SMAD4蛋白在撤栓后42 h卵巢组织中的表达量显著高于撤栓后18 h，其mRNA表达趋势及其编码蛋白表达趋势总体一致；而SMAD3基因mRNA及其编码蛋白的表达量在不同生理阶段卵巢组织中差异不显著。SMAD3基因mRNA及其编码蛋白在中卵泡中的表达量显著高于大卵泡；SMAD2和SMAD4基因表达量在不同直径卵泡中差异不显著。综上所述，SMAD2、SMAD3和SMAD4 基因在小尾寒羊卵巢周期性活动中的生物学功能存在差异，SMAD2和SMAD4 基因参与小尾寒羊发情初期至排卵前的卵巢生理变化，但不参与卵泡发育；而SMAD3参与卵泡发育。以上研究结果为进一步探索SMAD基因家族在小尾寒羊卵巢活动中的分子机理提供理论参考。

关键词: 小尾寒羊, 卵巢, 卵泡, SMAD2, SMAD3, SMAD4