Sohel Md Mahmodul Hasan, Hoelker Michael, Schellander Karl, Tesfaye Dawit
Department of Life Sciences, School of Environment and Life Sciences, Independent University, Bangladesh.
Animal Breeding and Husbandry Group, Institute of Animal Science, University of Bonn, Bonn, Germany.
Reprod Domest Anim. 2022 Oct;57(10):1208-1217. doi: 10.1111/rda.14195. Epub 2022 Jul 8.
Follicular fluid (FF) plays an important role during follicular development and it contains several bioactive molecules including extracellular microRNAs (ECmiRNAs) that may mediate cell-cell communication during follicular development. Yet, the distribution patterns of ECmiRNAs in FF is not well characterized. This study aims to investigate the distribution of ECmiRNAs in two major fractions, namely exosomal and non-exosomal, of bovine follicular fluid (bFF). Exosomal and non-exosomal fractions from bFF were separated using Exoquick™ exosomes precipitation kit. miRNA expression was evaluated using the human miRCURY LNA™ Universal RT miRNA PCR array system. Transmission electron microscopy and immunoblotting revealed that the isolated vesicles were exosomes. The real-time PCR-based expression analysis revealed that 516 miRNAs were detected in the exosomal fraction of bFF, while 393 miRNAs were detected in the non-exosomal fraction. Among the detected miRNAs, a total of 370 miRNAs were detected in both fractions, while 145 miRNAs and 23 miRNAs were solely detected in exosomal and non-exosomal fractions, respectively. Exploratory pathway analysis showed that the genes targeted by exosomal and non-exosomal miRNAs to be involved in MAPK, Wnt, FoxO, TGF-beta, Oxytocin, ErbB, PI3K-Akt, Neurotrophin signalling pathways which are believed to be involved in follicular development, cell proliferation, and meiotic resumption. The results of our study demonstrated that besides the exosomal fraction, non-exosomal fractions can carry a significant amount of miRNAs in bFF where the exosomal fraction carries a significantly higher number of detectable miRNAs.
卵泡液（FF）在卵泡发育过程中起着重要作用，它含有多种生物活性分子，包括细胞外微小RNA（ECmiRNAs），这些分子可能在卵泡发育过程中介导细胞间通讯。然而，卵泡液中ECmiRNAs的分布模式尚未得到充分表征。本研究旨在调查牛卵泡液（bFF）中两种主要组分，即外泌体和非外泌体组分中ECmiRNAs的分布情况。使用Exoquick™外泌体沉淀试剂盒分离bFF中的外泌体和非外泌体组分。使用人类miRCURY LNA™通用RT miRNA PCR阵列系统评估miRNA表达。透射电子显微镜和免疫印迹显示分离出的囊泡为外泌体。基于实时PCR的表达分析显示，在bFF的外泌体组分中检测到516种miRNA，而在非外泌体组分中检测到393种miRNA。在检测到的miRNA中，两个组分中共检测到370种miRNA，而分别在外泌体和非外泌体组分中仅检测到145种和23种miRNA。探索性通路分析表明，外泌体和非外泌体miRNA靶向的基因参与丝裂原活化蛋白激酶（MAPK）、Wnt、叉头框蛋白O（FoxO）、转化生长因子-β（TGF-β）、催产素、表皮生长因子受体（ErbB）、磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-Akt）、神经营养因子信号通路，这些通路被认为与卵泡发育、细胞增殖和减数分裂恢复有关。我们的研究结果表明，除了外泌体组分外，bFF中的非外泌体组分也可以携带大量的miRNA，其中外泌体组分携带的可检测miRNA数量明显更多。
