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.
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。