Department of Animal Reproduction, INIA-CSIC, Madrid, Spain.
Genetics and Functional Genomics, Clinic of Reproductive Medicine, Department for Farm Animals, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.
FASEB J. 2021 Dec;35(12):e22000. doi: 10.1096/fj.202101023R.
Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 10 EVs/ml and 6.0 × 10 EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p < .05). In UF-EVs, a total of 14 miRNAs were differentially abundant between stages. Greater differences were observed between stage 1 (S1) and stage 3 (S3), with 11 miRNAs enriched in S3 compared to S1. Functional enrichment analysis revealed the involvement of these miRNAs in relevant pathways such as cell signaling, intercellular junctions, and reproductive functions that may be implicated in oviduct and uterus modulation across the cycle, but also in their preparation for embryo/conceptus presence and development.
细胞外囊泡（EVs）存在于各种生物体液中，尤其是生殖液中，由于其在细胞间通讯中的可能作用而受到广泛关注。在 EVs 的不同生物活性分子 cargo 中，MicroRNAs（miRNAs）作为具有高临床潜力的有前途的诊断生物标志物而崭露头角。为了了解 EVs 在牛生殖道中的作用，我们旨在表征和分析输卵管液和子宫液（OF-EVs、UF-EVs）及其 miRNA 在发情周期中的特征。纳米颗粒跟踪分析和透射电子显微镜证实了 OF 和 UF 中存在小 EV 群体，（大小在 30 至 200nm 之间；浓度：OF 和 UF 分别为 3.4×10 EV/ml 和 6.0×10 EV/ml，与阶段无关）。EV 标志物（CD9、HSP70 和 ALIX 蛋白）的鉴定通过 Western blot 得到证实。miRNA 分析显示 OF-EVs 和 UF-EVs 中的 miRNA 丰度分别为 310 和 351 个。在 OF-EVs 中，有 9 个 miRNA 在周期的不同阶段存在差异丰度，其中 8 个 miRNA 从 S1 到 S4 呈递增趋势（p<.05）。在 UF-EVs 中，总共 14 个 miRNA 在不同阶段存在差异丰度。在 S1 和 S3 之间观察到更大的差异，与 S1 相比，S3 中 11 个 miRNA 富集。功能富集分析表明，这些 miRNA 参与了相关途径，如细胞信号转导、细胞间连接和生殖功能，这些途径可能与输卵管和子宫在周期中的调节有关，也可能与它们为胚胎/胚胎的存在和发育做准备有关。
