61 MicroRNA-148b secreted by bovine oviductal extracellular vesicles promotes embryo quality through TGF-β pathway

Extracellular vesicles (EVs) play an important role in cell-to-cell communication through their cargoes, especially microRNAs (miRNAs), which are small, noncoding RNAs regulating gene expression. In a previous study, we evidenced that bta-mir-148b was upregulated in EVs isolated from oviducal fluid (OF) of cyclic cows (Day 1 to 4, when the embryo is in the oviduct). In addition, bioinformatic analysis indicated that this miRNA is related to the TGF-β biological pathway, associated with blastocyst formation and embryo lineage segregation. Therefore, we aimed to evaluate the effect of miR-148b supplementation on bovine early embryo development and quality, and relative expression of selected genes coupled with the TGF-β pathway. Presumptive zygotes were cultured in SOF + 0.3% bovine serum albumin (BSA) (Control) or supplemented with 1 μM miR-148b mimics (miRCURY LNA miRNA, Qiagen) during: D1-D7 (miR148b) or D1-D4 (miR148b-OV) or D5-D7 (miR148b-UT) or 1 μM control mimics (miRCURY LNA miRNA mimic 5′FAM, Nº 339173, Qiagen). Embryos at ≥16-cell and D7 blastocysts (BD7) were collected and snap-frozen in LN₂ (3 pools: n = 10/group) to examine the relative expression of several transcripts linked to TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG [H2AFZ and 18S were used as housekeeping genes]) by RT-qPCR. To evaluate the total cell number, trophectoderm (TE), and inner cell mass (ICM) cells, BD7 (n = 30/group) were fixed, stained with anti-CDX2 and Hoechst 33342 and observed by a widefield fluorescence microscope. One-way ANOVA was used for all analyses. Blastocyst yield was not different between groups (Control: 24.4 ± 0.5, CM: 23.8 ± 0.5, miR148b: 24.2 ± 0.5, miR148b-OV: 24.4 ± 0.5 and miR148b-UT: 23.9 ± 0.6). Blastocysts’ total cell number, TE and ICM cells were higher (P ≤ 0.001) in miR148b (136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3, respectively) and miR148b-OV (135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, respectively) groups compared with the blastocysts from control (108.8 ± 1.0, 66.2 ± 0.7, 36.8 ± 1.0, respectively), CM (105.3 ± 1.2, 68.5 ± 1.1, 36.8 ± 0.8, respectively), and miR148b-UT (106.5 ± 1.1, 69.0 ± 0.9, 37.5 ± 1.2, respectively) groups. The ICM/TE ratio was equal for all groups (0.5 ± 0.0). The mRNA transcripts of SMAD1 and SMAD5 were significantly decreased (P ≤ 0.001) in 16-cell embryos and blastocysts from miR148b and miR148b-OV groups compared with the remaining groups, while TGFBR2 mRNA was only downregulated in 16-cell embryos and POU5F1 and NANOG mRNAs were upregulated (P ≤ 0.001) in blastocysts compared with the other groups. No differences were observed for SMAD2, SMAD3, BMPR2, and RPS6KB between groups. In conclusion, supplementation of bta-miR 148b mimics during the entire culture period (D1 to D7) or from D1 to D4 (when embryo is in the oviduct) improves embryo quality by acting in the TGF-β signalling pathway through changes in transcription of genes related to cellular differentiation and proliferation, highlighting the importance of the oviduct on embryo quality and development.

This research was supported by MINECO-Spain PID2019-111641RB-100, and K C-B was supported by a Maria Zambrano contract from European Union-NextGenerationEU.