Extracellular Vesicles in Luminal Fluid of the Ovine Uterus

Microvesicles and exosomes are nanoparticles released from cells and can contain small RNAs, mRNA and proteins that affect cells at distant sites. In sheep, endogenous beta retroviruses (enJSRVs) are expressed in the endometrial epithelia of the uterus and can be transferred to the conceptus trophectoderm. One potential mechanism of enJSRVs transfer from the uterus to the conceptus is via exosomes/microvesicles. Therefore, studies were conducted to evaluate exosomes in the uterine luminal fluid (ULF) of sheep. Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Transmission electron microscopy and nanoparticle tracking analysis found the isolates contained vesicles that ranged from 50 to 200 nm in diameter. The isolated extracellular vesicles were positive for two common markers of exosomes (CD63 and HSP70) by Western blot analysis. Proteins in the extracellular vesicles were determined by mass spectrometry and Western blot analysis. Extracellular vesicle RNA was analyzed for small RNAs by sequencing and enJSRVs RNA by RT-PCR. The ULF extracellular vesicles contained a large number of small RNAs and miRNAs including 81 conserved mature miRNAs. Cyclic and pregnant ULF extracellular vesicles contained enJSRVs env and gag RNAs that could be delivered to heterologous cells in vitro. These studies support the hypothesis that ULF extracellular vesicles can deliver enJSRVs RNA to the conceptus, which is important as enJSRVs regulate conceptus trophectoderm development. Importantly, these studies support the idea that extracellular vesicles containing select miRNAs, RNAs and proteins are present in the ULF and likely have a biological role in conceptus-endometrial interactions important for the establishment and maintenance of pregnancy.微囊泡与外泌体均为细胞释放的纳米颗粒，可携带小分子RNA、信使RNA（mRNA）与蛋白质，作用于远端靶细胞。在绵羊体内，内源性β逆转录病毒（enJSRVs）可在子宫内膜上皮细胞中表达，并可转移至胚胎滋养层。内源性β逆转录病毒从子宫向胚胎的潜在转移机制之一，便是通过外泌体/微囊泡实现。为此本研究针对绵羊子宫腔液（uterine luminal fluid, ULF）中的外泌体展开了相关探究。本研究采用ExoQuick-TC试剂盒，从发情周期第14天的未孕母羊与妊娠第14天的母羊子宫腔液中分离得到外泌体/微囊泡（下文统称为细胞外囊泡）。通过透射电子显微镜与纳米颗粒追踪分析发现，所分离得到的囊泡直径介于50至200纳米之间。经蛋白质印迹（Western blot）分析验证，所分离的细胞外囊泡呈现外泌体常见标志物CD63与热休克蛋白70（HSP70）阳性反应。本研究通过质谱分析法与蛋白质印迹检测，对细胞外囊泡内的蛋白质进行了鉴定。对细胞外囊泡内的RNA，本研究通过测序分析其小分子RNA组分，并通过逆转录聚合酶链反应（RT-PCR）检测内源性β逆转录病毒RNA。子宫腔液来源的细胞外囊泡中含有大量小分子RNA与微小RNA（miRNA），其中包含81种保守的成熟微小RNA。发情周期组与妊娠组母羊的子宫腔液细胞外囊泡中，均含有可在体外递送至异源细胞的内源性β逆转录病毒env与gag RNA。本研究结果支持如下假说：子宫腔液细胞外囊泡可将内源性β逆转录病毒RNA递送至胚胎，而这一过程对于内源性β逆转录病毒调控胚胎滋养层发育具有重要意义。尤为重要的是，本研究证实子宫腔液中存在携带有特定微小RNA、RNA与蛋白质的细胞外囊泡，且这类囊泡可能在胚胎-子宫内膜交互作用中发挥生物学功能，而该交互作用对于妊娠的建立与维持至关重要。