PDF(Pubmed)
Abstract：
BACKGROUND: Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-β pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality.
METHODS: Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses.
RESULTS: We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it\'s a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups.
CONCLUSIONS: Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-β signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.
METHODS: Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses.
RESULTS: We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it\'s a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups.
CONCLUSIONS: Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-β signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.
摘要：
背景：细胞外囊泡（EV）及其货物，包括MicroRNAs（miRNAs）在细胞间通讯中起着至关重要的作用。我们先前证明了循环奶牛输卵管液中bta-mir-148b的上调。该miRNA与细胞增殖中的TGF-β途径有关。我们的目的是验证miR-148b是否被胚胎接受，验证其目标基因，并探讨miR-148b对早期胚胎发育和质量的影响。
方法：在D1-D7（miR148b）或D1-D4（miR148b-OV：代表输卵管中的miRNA效应）或D4子宫-D7（miR148b-UT：代表在CMimD1中使用的miRNA效应）或1μM对照：收集≥16细胞和D7胚泡（BD7）的胚胎，以检查与TGF-β途径（TGFBR2，SMAD1，SMAD2，SMAD3，SMAD5，BMPR2，RPS6KB1，POU5F1，NANOG）相关转录本的mRNA丰度，总细胞数（TC），外胚层(TE),还评估了内细胞团(ICM)。单因素方差分析用于所有分析。
结果：我们证明miR-148b可以在16细胞胚胎和BD7中通过免疫染色被摄取，我们观察到SMAD5mRNA的减少，表明它是miR-148b的潜在靶标。卵裂率和囊胚率不受任何组的影响；然而，补充miR-148b模拟物对TC有积极作用，TE和ICM,miR148b的值为136.4±1.6、92.5±0.9、43.9±1.3，miR148b-OV组为135.3±1.5、92.6±1.2、42.7±0.8。此外，在miR148b和miR148b-OV组的16细胞胚胎和BD7中，SMAD1和SMAD5的mRNA转录本降低（P≤0.001），而POU5F1和NANOG在BD7中上调（P≤0.001），而TGFBR2仅在16细胞胚胎中下调。pSMAD1/5水平在miR148b和miR148b-OV组中较高。
结论：我们的发现表明，在整个培养期（D1-D7）或从D1-D4开始补充bta-miR-148b可以改善胚胎质量，并通过改变与细胞分化和增殖相关的基因的转录来影响TGF-β信号通路。这突出了miR-148b对胚胎质量和发育的重要性。
方法：在D1-D7（miR148b）或D1-D4（miR148b-OV：代表输卵管中的miRNA效应）或D4子宫-D7（miR148b-UT：代表在CMimD1中使用的miRNA效应）或1μM对照：收集≥16细胞和D7胚泡（BD7）的胚胎，以检查与TGF-β途径（TGFBR2，SMAD1，SMAD2，SMAD3，SMAD5，BMPR2，RPS6KB1，POU5F1，NANOG）相关转录本的mRNA丰度，总细胞数（TC），外胚层(TE),还评估了内细胞团(ICM)。单因素方差分析用于所有分析。
结果：我们证明miR-148b可以在16细胞胚胎和BD7中通过免疫染色被摄取，我们观察到SMAD5mRNA的减少，表明它是miR-148b的潜在靶标。卵裂率和囊胚率不受任何组的影响；然而，补充miR-148b模拟物对TC有积极作用，TE和ICM,miR148b的值为136.4±1.6、92.5±0.9、43.9±1.3，miR148b-OV组为135.3±1.5、92.6±1.2、42.7±0.8。此外，在miR148b和miR148b-OV组的16细胞胚胎和BD7中，SMAD1和SMAD5的mRNA转录本降低（P≤0.001），而POU5F1和NANOG在BD7中上调（P≤0.001），而TGFBR2仅在16细胞胚胎中下调。pSMAD1/5水平在miR148b和miR148b-OV组中较高。
结论：我们的发现表明，在整个培养期（D1-D7）或从D1-D4开始补充bta-miR-148b可以改善胚胎质量，并通过改变与细胞分化和增殖相关的基因的转录来影响TGF-β信号通路。这突出了miR-148b对胚胎质量和发育的重要性。
