Analyses of circRNA profiling during the development from pre-receptive to receptive phases in the goat endometrium

Background: Recent studies have revealed that noncoding RNAs play important regulatory roles in the formation of endometrial receptivity. Circular RNAs(circRNAs) are a universally expressed noncoding RNA species that have been recently proposed to act as miRNA sponges that directly regulate expression of target genes or parental genes.Results: We used Illumina Solexa technology to analyze the expression profiles of circRNAs in the endometrium from three goats at gestational day 5(pre-receptive endometrium, PE) and three goats at gestational day 15(receptive endometrium, RE). Overall, 21,813 circRNAs were identified, of which 5,925 circRNAs were specific to the RE and 9,078 were specific to the PE, which suggested high stage-specificity. Further analysis found 334 differentially expressed circRNAs in the RE compared with PE(P 0.05). The analysis of the circRNA-miRNA interaction network further supported the idea that circRNAs act as miRNA sponges to regulate gene expression.Moreover, some circRNAs were regulated by estrogen(E2)/progesterone(P4) in endometrial epithelium cell lines(EECs) and endometrial stromal cell line(ESCs), and each circRNA molecule exhibited unique regulation characteristics with respect to E2 and P4.Conclusions: These data provide an endometrium circRNA expression atlas corresponding to the biology of the goat receptive endometrium during embryo implantation.     

UFMylation is associated with LPS-induced inflammatory response in goat endometrial epithelial cells

The endometrium plays an important role in the defence against invading pathogens, although the mechanisms are not clear. UFMylation is a recently discovered novel ubiquitination-like modification system that plays a pivotal role in inflammation and the immune response. The purpose of this study was to investigate the effects of UFMylation on lipopolysaccharide (LPS)-induced inflammatory responses in immortalized goat endometrial epithelial cells (gEECs). Ubiquitin-fold modifier conjugating enzyme 1 (UFM1) and DDRGK domain containing 1 (DDRGK1) were mainly localized in the luminal epithelium and glandular epithelium of mouse and goat endometrial tissues. The expression levels of UFM1, ubiquitin-like modifier activating enzyme 5 (UBA5), UFM1 specific ligase 1 (UFL1) and DDRGK1, as key components of the UFMylation system, were significantly activated by 5 μg/mL LPS-induced inflammatory response in gEECs for 6 hr. Meanwhile, the expression levels of interleukin-6 (IL-6) were significantly upregulated, and tumour necrosis factor-α (TNF-α) was significantly down-regulated after overexpression of UFM1 in gEECs. Additionally, we observed UFM1 and DDRGK1 were markedly increased on LPS-stimulated mouse endometritis in vivo. In conclusion, the current study demonstrated that UFMylation was significantly activated by LPS and might be involved in regulating inflammatory response in gEECs.     

精氨酸对干扰素-tau处理牛子宫内膜上皮细胞基因表达的影响

试验旨在研究精氨酸（Arg）对干扰素-tau（interferon-tau,IFNT）处理的牛子宫内膜上皮细胞的调控作用及其可能的机制。将牛子宫内膜上皮细胞接种至6孔细胞培养板中,当达到70%~80%融合度时,向细胞培养基中加入不同浓度的IFNT（0、100 ng/mL）,12 h后收集细胞检测未折叠蛋白反应（unfolded protein response,UPR）通路相关基因的表达情况;用不同浓度（0、0.05、0.1、0.2、0.5、1、2 mmol/L）Arg处理牛子宫内膜上皮细胞,24 h后用CCK8法检测其对增殖率的影响,筛选出Arg的最佳作用浓度;将子宫内膜上皮细胞随机分为Arg饥饿组、Arg饥饿+IFNT处理组、Arg添加+IFNT处理组,在Arg饥饿或添加处理6 h后加入100 ng/mL IFNT,IFNT处理12 h后检测UPR通路（BiP、PERK、CHOP、ATF6）、凋亡（BAX、Caspase9）和容受性（HOXA10）相关基因表达情况。结果显示,IFNT刺激可诱导牛子宫内膜上皮细胞UPR通路相关基因BiP、PERK、CHOP、ATF6的表达显著上调;0.5 mmol/L Arg可显著提高牛子宫内膜上皮细胞的增殖率（P<0.05）;Arg缺失可显著上调IFNT诱导下牛子宫内膜上皮细胞中BiP、PERK、CHOP、ATF6和BAX基因mRNA的表达（P<0.05）,并显著下调HOXA10基因mRNA的表达（P<0.05）,但对Caspase9基因mRNA表达无显著影响（P>0.05）。结果表明,Arg缺失可导致牛子宫内膜上皮细胞发生内质网应激,并影响子宫内膜宫受性的建立,可为进一步揭示Arg对牛子宫内膜的调控机制及制定减少妊娠早期胚胎丢失的营养调控策略提供参考。     

siRNA抑制牛子宫内膜上皮细胞Nrf2基因的表达研究

为探讨核因子E2相关因子（nuclear factor E2 related factor，Nrf2）基因沉默及激活后对牛子宫内膜上皮细胞的影响，本试验利用小分子干扰（siRNA）技术及Nrf2的激动剂叔丁基对苯二酚（tBHQ），分别从Nrf2基因的下调和上调表达来研究Nrf2对牛子宫内膜上皮细胞中血红素加氧酶-1（HO-1）和Homebox A10（HOXA10）基因表达的影响。结果显示，经实时荧光定量PCR方法检测，在设计的2条siRNA序列（siRNA-1209和siRNA-1672）中，siRNA-1672在终浓度为75 nmol/L、作用时间24 h时抑制效果较好，抑制效率在80%以上；经Western blotting方法检测，Nrf2蛋白表达水平在转染后96 h极显著下降（P<0.01），而HO-1和HOXA10的mRNA表达量分别下降了60%和70%（P<0.01），蛋白表达量在96 h后极显著或显著下降（P<0.01；P<0.05）。此外，经CCK8方法检测，Nrf2基因表达沉默后，牛子宫内膜上皮细胞增殖能力减弱。而在tBHQ激活Nrf2试验中，经Western blotting方法检测，tBHQ终浓度为30 μmol/L时Nrf2蛋白表达量最高，极显著高于对照组（P<0.01），且HO-1和HOXA10的蛋白表达量与对照组相比也明显上升。结果表明，本试验设计的siRNA-1672能特异性地抑制牛子宫内膜上皮细胞Nrf2的表达，而抑制Nrf2的表达会导致牛子宫内膜上皮细胞增殖能力下降，且Nrf2对牛子宫内膜上皮细胞HO-1和HOXA10基因表达存在调控作用。     

YAP1在发情期不同繁殖力湖羊子宫内膜中的表达模式及功能分析

旨在分析Hippo信号通路的效应基因YAP1在不同繁殖力湖羊子宫内膜中的表达模式及功能。本研究根据系谱档案和BMPR-1B基因多态性分析，将9只体况相近且健康的经产母羊分为3组(HBB、LBB以及LB+,n=3)。提取不同繁殖力湖羊子宫内膜组织RNA,并反转录为cDNA,实时荧光定量PCR (RT-qPCR)分析Hippo信号通路中几个主效基因的表达模式。运用生物信息学方法分析YAP1在不同物种间的氨基酸同源性及其亲缘关系。体外分离湖羊子宫内膜基质细胞，利用RNA干扰和细胞转染技术体外干扰YAP1,采用实时荧光定量PCR (RT-qPCR)分析YAP1基因对子宫容受性相关基因的影响。利用流式细胞术分析干扰YAP1对细胞凋亡的影响，同时利用RT-qPCR和Western blot检测干扰YAP1后凋亡及线粒体相关基因的表达。结果显示，Hippo信号通路中YAP1、LATS1、MST1基因在不同繁殖力湖羊子宫内膜组织中的表达水平存在差异，其中YAP1基因在高繁殖力(HBB)湖羊子宫内膜中的表达量显著高于两组低繁殖力(LB+、LBB)湖羊；氨基酸同源性及系统进化树分析显示，绵羊YAP1蛋白与...     

山羊CREBZF蛋白两种亚型过表达载体的构建及其对子宫内膜上皮细胞凋亡的影响

旨在构建编码山羊CREBZF蛋白2种亚型(长亚型SM ILE和短亚型Zhangfei)基因的过表达载体,并初步探究CREBZF对山羊子宫内膜上皮细胞(gEECs)凋亡的影响.本研究针对编码山羊CREBZF不同亚型的基因CDS区分别设计引物,分段进行PCR扩增;运用无缝克隆技术将所获得各目的 基因片段与线性化pcDNA3...     

Concentration effect of prostaglandin E2 on the growth factor expression and cell proliferation in bovine endometrial explants and their kinetic characteristics

Bovine endometrium undergoes various physiological and histological changes that are necessary for blastocyst implantation during oestrous cycle. From pro‐oestrus to late‐oestrus, endometrium thickens gradually for implantation preparation and exhibits remarkable capacity for self‐repair after uterine lining shedding while implantation does not occur. The prostaglandin E₂ (PGE ₂) secretion pattern is synchronized with endometrial growth during oestrous cycles in bovine endometrium; however, limited information is available regarding the association between PGE ₂ secretion and endometrial growth. In this study, the concentration (10^?9 to 10^?5 M) and time effect (2–36 hr) of PGE ₂ treatment on a series of growth factors are essential for endometrial growth including connective tissue growth factor (CTGF ), fibroblast growth factor‐2 (FGF ‐2), interleukin‐8 (IL ‐8), transforming growth factor‐β1 (TGF ‐β1), matrix metalloproteinase‐2 (MMP ‐2), and vascular endothelial growth factor A (VEGFA ) mRNA and protein expression, and proliferation of epithelial and fibroblast cells was investigated in bovine endometrial explants in vitro. The results indicated that PGE ₂ at concentration about 10^?7 to 10^?5 M could up‐regulate CTGF , FGF ‐2, IL ‐8, MMP ‐2, TGF ‐β1, VEGFA mRNA and protein expression, and could induce the proliferation of epithelial and fibroblast cells and reduce the proapoptotic factor (caspase‐3) expression in bovine endometrial explants in vitro. These results collectively improved the possibility of PGE ₂ functions in endometrial growth during oestrous cycles.     

黄藤素对山羊子宫内膜上皮细胞的体外毒性研究

为了探究黄藤素的体外细胞毒性,采用不同浓度的黄藤素作用山羊子宫内膜上皮细胞（EECs）后,采用MTT法绘制细胞生长曲线;倒置显微镜下观察细胞生长状态;瑞氏-姬姆萨染色法观察细胞形态学变化并通过测定乳酸脱氢酶（LDH）含量检测细胞膜的完整性。结果显示,当黄藤素的浓度为10~227 μg/mL时,黄藤素对EECs有促增殖的作用;当浓度大于227 μg/mL时出现细胞增殖抑制现象,药物对细胞的半数增殖抑制率（IC₅₀）为360 μg/mL;瑞氏-姬姆萨染色结果显示,100、200 μg/mL的黄藤素作用于EECs时均未对其细胞核、细胞浆的形态产生影响;LDH结果显示,药物浓度≥250 μg/mL时细胞LDH的释放量极显著高于正常细胞组（P<0.01）。综上所述,黄藤素作用于山羊EECs后呈现一定的毒性作用,且呈现显著的剂量依赖效应,作用于EECs的安全剂量范围为≤250 μg/mL。     

Development of an in vitro model for the analysis of bovine endometrium using simple techniques

This study aimed to develop an in vitro model for the analysis of the bovine endometrium. Immunofluorescent staining revealed that the hetero‐spheroids and the cultured explants showed almost similar structure in the localization of bovine endometrial epithelial cells and endometrial stromal cells, except the glandular‐like structure of the epithelial cells inside the explants. Gelatin zymography revealed that the hetero‐spheroids did not express matrix metalloproteinases (MMPs) after 4 days of culture, but strong MMP expressions were observed in the cultured explants until 7 days of culture. Additionally, expression of progesterone receptor (PR), estrogen receptor alpha (ERα), type I interferon receptor 1 (IFNAR1) and 2 (IFNAR2) messenger RNA was observed both in the homo‐ and hetero‐spheroids. The expression of oxytocin receptor (OTR) mRNA in E2 and E2+P4 (1,3,5(10)‐Estratrien‐3, 17β‐diol + 4‐Pregnen‐3, 20‐dinone) treated groups were significantly (P < 0.05) higher than that of the control group of spheroids. In case of cultured explants, the expression of PR and OTR mRNA were significantly (P < 0.05) higher in E2 treated groups compared to the control groups. Hepatocyte growth factor (HGF) mRNA expression was also higher in P4 treated groups at 10 days in culture (P < 0.05). In a nutshell, the in vitro model developed in this study for the analysis of the endometrium may provide a new platform for extensive research on bovine endometrial function.     

The Study on the Cytotoxicity of Palmatine on Goat Endometrial Epithelial Cells in vitro

To explore the cytotoxicity of palmatine in vitro,goat endometrial epithelial cells (EECs) were stimulated by different concentrations of palmatine,then the growth curve of cell was drew by MTT assay which could detect the cell proliferation. The cell growth state was observed by inverted microscope,and Wright-Giemsa staining was used to observe morphological changes of the cells while LDH test was used to check the membrane permeability of EECs. The results showed that when the concentration was 10 to 227 μg/mL,palmatine had a role in promoting proliferation of the EECs,while when the concentration was more than 227 μg/mL,cell proliferation was inhibited,and 50% inhibitory rate of proliferation (IC₅₀) of this drug on EECs was 360 μg/mL.Wright-Giemsa staining results showed that themorphology of the nucleus and cytolymph were not significantly changed when 100 and 200 μg/mL palmatine effected EECs. LDH results showed that when the palmatine concentration was more than 250 μg/mL,LDH content was extremely significantly higher than that of normal cell group(P<0.01).In conclusion,palmatine presented certain toxic effect on EECs with a significant dose-response effect,and the safe dose range of palmatine was less than 250 μg/mL.     

EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中血管内皮生长因子基因表达的影响

[目的]观察EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中血管内皮生长因子(vascular endothelial growth factor,VEGF)基因表达的影响,探讨前列腺素类化合物对奶牛子宫内膜组织修复的机制。[方法]分离培养奶牛子宫内膜上皮细胞,采用荧光定量PCR技术检测EP2受体激动剂Butaprost对奶牛子宫内膜上皮细胞中VEGFmRNA表达的影响。[结果]10-6mol/L的Butaprost作用于奶牛子宫内膜上皮细胞4、8、16、24h可显著(P<0.05)或极显著(P<0.01)地促进VEGFmRNA的表达。[结论]EP2受体激动剂Butaprost能够促进奶牛子宫内膜上皮细胞中VEGF基因的表达。     

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0 = day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E₂) were detected in day 25 conceptuses. Concentrations of E₂ were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E₂ and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E₂ and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.     

山羊子宫内膜上皮细胞转染pCI-neo-hTERT质粒后的永生化

为获得大量的、具有高度同一性和表型功能正常的山羊子宫内膜上皮细胞(EEC),本试验将含人端粒酶逆转录酶(hTERT)基因的真核表达质粒pCI-neo-hT ERT导入原代山羊EEC中,筛选稳定转染的细胞克隆,进行表型鉴定,利用免疫细胞化学方法检测hTERT导入后细胞端粒酶活性的表达情况,探讨转染后细胞的生长特性。结果显示,转染后获得的阳性克隆细胞及其传代细胞呈明显铺路石状,与原代细胞形态一致,具有接触抑制性;角蛋白检测阳性;细胞具有较高的端粒酶活性,目前已传至50代仍稳定增殖;100nmol/L的雌二醇(E2)可促进该细胞的增殖;50,100nmol/L的孕酮(P4)可明显抑制该细胞的增殖。这表明hTERT导入山羊EEC后,可使其重建端粒酶活性,从而获得大量的、具有高度同一性和表型正常的细胞。