89 Impact of bta-mir-483-3p carried within oviductal fluid’s extracellular vesicles of pregnant cows on in vitro embryo development and quality

MicroRNAs (miRNAs) modulate gene expression and signaling pathways involved in early embryo development via post-transcriptional processes. They are selectively loaded into extracellular vesicles (EVs) and participate in maternal-embryonic communication. Bta-mir-483–3p was uniquely found in oviducal fluid EVs from pregnant cows compared to nonpregnant (Mazzarella et al. 2021 Front. Vet. Sci. 8, 639752). We aimed to assess bta-miR-483–3p’s impact on in vitro early embryo development and quality. In vitro-produced presumptive zygotes (n = 1108) cultured in SOF+0.3% BSA (control) or SOF + 0.3% BSA supplemented with 1 μM of miR-483–3p mimic (miR483–3p), or 1 μM of miR-483–3p control mimic (CM), or 1 μM of its inhibitor (Inh483–3p), or 1 μM of control inhibitor (CInh), in different time frames based on the embryo physiological location in the reproductive tract: (1) Days 1–4: miR483–3p/OV or Inh483–3p/OV, representing miRNA effect in the oviduct; (2) Days 4–7: miR483–3p/UT or Inh483–3p/UT, representing miRNA effect in the uterus; or (3) Days 1–7: miR483–3p, Inh483–3p, CM or control. MiR483–3p mimic, its inhibitor, and their controls were purchased from Qiagen’s miRCURY LNA miRNA line. Developmental rates were evaluated on Day 4 (≥16-cell stage, 16C) and Day 7 (blastocyst stage, BD7). Embryos from both stages were collected to assess their quality, by evaluating their mitochondrial activity (Mito Tracker Deep Red) and lipid content (Bodipy). One-way ANOVA and Tukey test were used for all comparisons. At Day 4, the proportion of 16C embryos was lower (P < 0.05) in Inh483–3p, Inh483–3p/OV, and CInh groups compared to the others. At Day 7, blastocyst yield was higher (P < 0.05) in control: 25.4 ± 0.7%, CM: 25.6 ± 0.6%, miR483–3p: 25 ± 0.9%, miR483–3p/OV: 24.6 ± 0.3% and miR483–3p/UT: 24 ± 0.9%, compared to CInh: 15.2 ± 1.3%, Inh483–3p: 14.7 ± 0.5%, Inh483–3p/OV: 14.2 ± 0.7% and Inh483–3p/UT: 14.6 ± 1.3%. No significant differences were observed in mitochondrial activity among 16C embryos, while the miR483–3p and miR483–3p/OV groups exhibited lower (P < 0.05) lipid droplet content. Blastocyst stage BD7 from miR483–3p, miR483–3p/OV, and miR483–3p/UT groups exhibited higher (P < 0.05) mitochondrial activity from all other groups, while BD7 from control and CM groups had higher activity (P < 0.05) compared to CInh, Inh483–3p, Inh483–3p/OV, and Inh483–3p/UT groups. On the other hand, lipid content was significantly decreased in BD7 from miR483–3p, miR483–3p/OV, and miR483–3p/UT compared to all other groups, while those from control and CM groups exhibited lower (P < 0.05) lipid content compared to CInh, Inh483–3p, Inh483–3p/OV, and Inh483–3p/UT groups. In conclusion, although the effect of Inh483–3p and CInh groups requires further investigation, the results highlight the potential impact of miR483–3p mimic on embryo quality when added to in vitro culture by increasing blastocysts’ mitochondrial activity and decreasing their lipid content, suggesting its role in pre-implantation embryo-maternal interaction.

This study was supported by Spanish MICIN: PID2019–111641RB-I00, Maria Zambrano (KCB) and Margarita Salas (YNC).