UC Davis

Recent reports of bovine embryonic stem cell (bESC) establishment and in vitro gametogenesis (IVG) in the mouse open the door to recapitulate oogenesis from bESCs in vitro, however little progress has been made toward this feat. Critical gaps remain in our understanding of bovine germ cell development, which will be necessary to establish a system that supports efficient in vitro germline formation as well as to assess the resulting cells once obtained. Additionally, progress in other models has shown the importance of pluripotent state on cellular capacity for in vitro germline induction. Therefore, the goal of this dissertation was to shed light on the requirements of livestock pluripotency and germline development to set the basis to establish reliable bovine in vitro oogenesis systems.We first examined the pluripotent state of bESC lines and explored the potential to stabilize these cells in intermediate states of pluripotency, due to the limitations of primed ESCs for germline induction. We found that regardless of the initial blastocyst stage that bESCs were derived from, cell lines exhibited similar transcriptional and functional characteristics of primed pluripotency. However, the demonstration that bESCs could be efficiently established from early blastocysts increases the pool of embryos that may be targeted for bESC derivation approaches. We also investigated alternative formative media for bESC establishment, but found that conditions that supported this state in other mammalian species did not translate to cattle. This highlights the unique signaling requirements of bovine pluripotency and the necessity of further studies to establish alternative conditions for bESCs with high germline competence. Specification of primordial germ cells (PGCs), the precursors of oocytes and sperm, occurs during early embryonic development. The first step of IVG is induction of ESCs into PGC-like cells. Despite wide interest in replicating this event in vitro, little was known about PGC specification in the cow. We identified founder PGCs in the posterior epiblast of bovine embryos at day 16, by unique co-expression of conserved early germline proteins SOX17, PRDM1, and TFAP2C, with absent SOX2. We found that PGCs had initiated migration by day 20, at which point they were distributed through the allantois and hindgut endoderm, but were not yet highly proliferative. Single-cell transcriptome analysis of bovine embryos at day 20-22 allowed us to capture a small population of bovine PGCs, and gave insights into early germline development, including acquisition of the germ cell program, repression of somatic marks, and initiation of epigenetic remodeling. Further, we identified candidate cell surface markers for sorting PGCs – or in vitro-induced counterparts – for future experiments. This work provides the earliest observation of PGCs in cattle and demonstrates a conserved program in bovine germline emergence with other species that form as an embryonic disc. We continued from studying PGC specification and migration onset in the embryo through the advanced development of these cells into oocytes in the fetal ovary. Expanding upon a report from day 50, we traced the bovine germline by single-cell RNA-sequencing at days 70, 90, and 120 of fetal ovarian development. This revealed the transcriptional profile of bovine germ cells from gonadal PGCs through germline commitment into oogonia, initiation of meiosis, arrest at prophase I, and development of primordial oocytes within follicles, as well as cell surface marker candidates for each of these stages. Further, we explored the molecular profile of the somatic niche of the ovary and the interactions of these cells with the germline, which gives evidence for signals that could support germ cell development in a culture dish. In conclusion, this dissertation provides detailed and novel insights into bovine pluripotency and germline establishment. The knowledge gained from these studies may help in the development of assisted reproductive technologies of livestock, humans, and endangered ungulates. Moreover, these studies highlight the cow as a valuable model of human reproductive development and offer important context of embryonic/fetal events that cannot be directly studied in humans.