Rio Paul Del, Madan Pavneesh
Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
Front Vet Sci. 2021 Apr 8;8:658968. doi: 10.3389/fvets.2021.658968. eCollection 2021.
Distinct miRNA populations have been detected in the spent media of culture systems. However, profiling has been limited to media conditioned with blastocyst-stage embryos. Therefore, the aim of the study was to profile extracellular miRNAs throughout the pre-implantation period in bovine embryos. To achieve this, cumulus oocyte complexes were aspirated from ovaries, matured, fertilized, and cultured under standard laboratory procedures to the 2-cell, 8-cell, or blastocyst stage of development. At each developmental stage, 25 μl of spent culture media was collected, pooled to 300 μl, and processed for total RNA extraction. culture media, which never came in contact with any embryos, were additionally processed for total RNA extraction to serve as a negative control. Following hybridization on a GeneChip miRNA 4.0 array, comparative analysis was conducted between spent media and control samples. In total, 111 miRNAs were detected in the spent media samples, with 56 miRNAs identified in blastocyst spent media, 14 miRNAs shared between 8-cell and blastocyst spent media, 7 miRNAs shared between all 3 conditions, and 6 miRNAs exclusive to 2-cell spent media. miRNA-mRNA target prediction analysis revealed that the majority of genes predicted to be regulated by the miRNAs identified in the study have roles in cellular process, metabolic process, and biological regulation. Overall, the study suggest that miRNAs can be detected in the spent media of culture system throughout the pre-implantation period and the detected miRNAs may influence genes impacting early embryo development.
在培养系统的废弃培养基中已检测到不同的miRNA群体。然而，分析仅限于用囊胚期胚胎处理过的培养基。因此，本研究的目的是分析牛胚胎植入前整个时期的细胞外miRNA。为实现这一目标，从卵巢中吸出卵丘卵母细胞复合体，在标准实验室程序下进行成熟、受精，并培养至2细胞、8细胞或囊胚发育阶段。在每个发育阶段，收集25μl废弃培养基，汇集至300μl，然后进行总RNA提取处理。从未与任何胚胎接触过的培养基也另外进行总RNA提取处理，用作阴性对照。在GeneChip miRNA 4.0阵列上进行杂交后，对废弃培养基和对照样品进行了比较分析。在废弃培养基样品中总共检测到111种miRNA，其中56种miRNA在囊胚废弃培养基中被鉴定出来，14种miRNA在8细胞和囊胚废弃培养基中共有，7种miRNA在所有三种条件下都有，6种miRNA是2细胞废弃培养基所特有的。miRNA - mRNA靶标预测分析表明，预测受本研究中鉴定出的miRNA调控的大多数基因在细胞过程、代谢过程和生物调节中发挥作用。总体而言，该研究表明在植入前整个时期的培养系统废弃培养基中均可检测到miRNA，且检测到的miRNA可能影响影响早期胚胎发育的基因。
