92 A single cell atlas of bovine peri-implantation embryo development

In bovines, a viable embryo will enter a critical two- to three-week period of embryogenesis when the blastocyst undergoes extensive cellular proliferation and changes from a spherical shape to an elongated, filamentous form in preparation for implantation. Failure of development during this time represents one of the major causes of early pregnancy loss. However, our understanding of molecular phenotypes during this process is extremely limited. Here, we characterised the single cell transcriptional profile of bovine pre- and peri-implantation embryos at day 8, 12, 14, 16, and 18 after fertilisation. Different stages of bovine embryos were collected by a standard nonsurgical flush following a superovulation and timed artificial insemination protocol. Single cells at each developmental stage (Day 12 [d12]: n = 6; Day 14 [d14]: n = 2; Day 16 [d16]: n = 3; Day 18 [d18]: n = 1) were isolated and captured by the 10X Chromium Controller. Single cell cDNA libraries were prepared using Chromium Single Cell 3′ V3 Reagent Kit (10x Genomics Inc.). Libraries were processed by multiplexing and sequencing using the Illumina NovaSEqn 6000 System. An average of 11,000 cells per stage were analysed. Single-cell analysis of Day 8 blastocysts (n = 20) was performed using SMART-seq v4 protocol (Clontech). Data analyses were performed using the CellRanger version 3.0 pipeline (10x Genomics Inc.) and an R package (Seurat version 4.0). We were able to map the transcriptome trajectories of different cell lineages during bovine peri-implantation. In a Day 8 blastocyst, we captured all three lineages, including epiblast, hypoblast, and trophectoderm cells. The embryos underwent significant elongation in size in Day 12, 14, 16, and 18 embryos. At molecular level, our analysis revealed that the epiblast first differentiates into ectoderm at Day 12 and 14, and subsequently develops to mesoderm, endoderm, and ectoderm at Day 16 and 18. We also observed that the hypoblast differentiates into extraembryonic endoderm and mesoderm, parietal hypoblast, and yolk sac from Day 12 to Day 18. Finally, we found different types of trophoblasts based on the gene expression patterns, reflecting the very dynamic process of trophoblast lineage differentiation during peri-implantation period. Two major observations were found in the trophoblast development, with one develops into trophoblast sub-lineages as defined by the uninuclear and binuclear trophoblast marker gene expression at Day 18 embryos, and the other develops into two different types of trophoblasts at embryos from Day 12 and Day 18 as revealed by the clustering analysis, presumably the polar and mural trophoblast cells. We have also identified novel marker genes associated with each cell lineage subsequently emerge during bovine peri-implantation development. Together, these analyses generate comprehensive atlases of early bovine embryo development, which will serve as a gold standard reference for the assessment of cell identities and molecular characteristics of lineage development in bovine peri-implantation.