91 Invivo- and invitro-produced bovine embryos have different microRNA profiles after invitro individual culture

Abstract

Invivo- and invitro-produced bovine embryos have different metabolic characteristics, embryonic development, and gene transcription. Additionally, pregnancy rates at 30 days (on average 51% and 34% when using fixed-time AI and invitro production, respectively) are different in beef cattle. Between Days 8 and 17 of the oestrous cycle, concurrent with embryo-maternal recognition, is when 40% of embryonic losses occur. These losses may occur due to altered embryo-maternal cross-talk. MicroRNA (miRNA) can be involved in this communication; however, its potentially regulated pathways in invivo and invitro embryos on Day 9 are unknown. Our hypothesis is that bovine embryos produced invivo and invitro contain different miRNA profiles, even after invivo bovine embryo were invitro cultured. Cows had the follicular wave synchronized and were superovulated to produce invivo or invitro bovine embryos. For the invitro group, on Day −8 of the protocol, the dominant follicles were recovered by ovum pickup, and invitro embryo production was performed to obtain embryos. For the invivo group, on Day −8, the cows were inseminated 12 and 24h after GnRH analogue application and on Day 7 after expected oestrus, uterine flushing was performed to obtain the embryos. Embryos from both groups were individually cultured for 48h. Three pools (of 5 embryos each) per group were used for reverse transcription of miRNAs from total RNA using miScript II RT Kit (Qiagen). Relative levels of 383 bovine miRNAs were determined using the geometric mean of miR-99b, RNU43 snoRNA, and Hm/Ms/Rt U1 snRNA by RT-qPCR. Differences in relative levels of miRNAs were determined by Student's t-test. A total of 210 miRNAs were detected in invivo and invitro embryos, and 13 out of 210 were differently identified between the groups. In invivo embryos, 6 miRNAs were up-regulated, whereas 7 miRNAs were up-regulated in invitro embryos. TARGETSCAN software was used to identify genes predicted as modulated by each miRNA. The top 100 genes predicted were used to identify enriched pathways according to DAVID Bioinformatics Resources. The miRNAs (miR-129, miR-132, miR-155, miR-192, miR-215, and miR-377) up-regulated in invivo embryos modulated pathways that include signaling pathways regulating pluripotency of stem cells (16 genes), TGF-β (11), hippo (10), oestrogen (8), and cell cycle (7). Moreover, miR-23a, miR-338, miR-34a, miR-491, miR-92b, miR-940, and miR-1271, which were increased in invitro embryos, regulate PI3K-Akt (17 genes), signaling pathways regulating pluripotency of stem cells (10), oestrogen (9), toll-like receptor (9), Wnt (9), and HIF-1 (7). The results demonstrate that even after 48h of invitro culture, bovine embryos produced invivo and invitro have different miRNA profiles that modulate pathways associated with embryonic development on Day 9. Furthermore, these results suggest that bioactive molecules, such as miRNAs, can modify embryo-maternal cross-talk, depending on the environment where the embryos are produced. Funding was provided by FAPESP 2017/19681-9, 2014/22887-0, and 2018/13155-6.