Degree

Doctor of Philosophy (PhD)

Department

School of animal science

Document Type

Dissertation

Abstract

Embryo loss and early pregnancy failure are major causes of infertility in cattle. However, most of the molecular and cellular mechanisms that orchestrate trophoblast development and pregnancy establishment remain unknown, due to difficulties in conducting in vivo experiments in cattle and lack of in vitro cell models. This dissertation provides two unique platforms for studying bovine early placental trophoblast development and early embryogenesis.

First, we have identified that an extended pluripotent stem cells (EPSCs) culture condition, LCDM (hLIF, CHIR99021, DiM and MiH), is effective in supporting long-term (>70) passage of bovine trophoblast stem cells (bTSCs) from blastocysts. Bovine TSCs highly express TE-related markers (CDX2, GATA3, and KRT8) but not SOX2. Next, we have demonstrated the ability of bTSCs to generate mature trophoblast cells in vitro and in vivo. Third, we have conducted epigenomic analysis of bTSCs, bESCs, and trophectoderms of different stage of embryos via assay for transposase-accessible chromatin using sequencing (ATACseq) and whole-genome bisulfite sequencing (WGBS), and defined epigenomic features (chromatin accessibility and DNA methylation) of bTSCs. Finally, we have elucidated important regulators and signaling pathways administering bovine early placental development.

Second, we have developed a 3D culture system to generate bovine blastocyst-like structures (blastoids) through assembling bovine EPSCs and TSCs. First, we have optimized a culture condition that could support the efficient formation of bovine blastoids. Bovine blastoids resemble blastocysts, containing a blastocele-like cavity, an outer trophectoderm and an inner cell mass-like compartment. Second, we have performed immunofluorescence analysis and found that bovine blastoids consist of cells with characteristic of epiblast (SOX2), hypoblast (SOX17) and trophectoderm (GATA3 and CDX2). Third, we have conducted 3D suspension culture of bovine blastoids and evaluated the in vitro prolonged growth of blastoids. More importantly, we have transferred blastoids into recipients and identified that blastoids have the capacity to trigger signaling of maternal recognition of pregnancy. Finally, a comprehensive single cell RNA sequencing for blastoids and blastocysts has shown that blastoids contain all three cell lineages and transcriptionally resembled day 7.5 blastocysts.

Thus, bovine TSCs and blastoids are faithful and scalable models for investigating bovine placental development and early embryo development, respectively.

Date

6-30-2023

Committee Chair

Jiang, Zongliang

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