139 Transcriptome analysis of mouse embryos developed from in vitro follicle culture-derived oocytes provides insights into transcriptional dysregulation at the cleavage stage

In vitro follicle culture (IVFC) is an emerging technique that allows for the growth and development of the oocyte within the follicle in a culture system. Yet it is unclear how the IVFC period affects the oocyte and subsequent embryonic molecular identity. Importantly, many embryos developed from IVFC-derived oocytes arrest at the early cleavage stages. However, it is unknown whether these embryos navigate the key developmental event of zygotic genome activation (ZGA), in which transcription begins for the first time in the preimplantation embryo. We hypothesized that ZGA might be impaired in embryos developed from IVFC-derived oocytes, which arrest at the 2-cell stage when ZGA normally occurs in mice. To test this, we performed RNA-seq to map 2-cell stage embryo transcriptome identities. We performed IVFC using secondary follicles from BDF1 female Day 12 pups (Jin et al. 2010 Fertil. Steril. 93.8, 2633–2639). The follicles were cultured in alginate beads (to preserve the three-dimensional structure) until the late antral stage, and then the IVFC-derived oocytes were matured in vitro and chemically activated to generate parthenote 2-cell stage embryos. Mature oocytes as well as parthenote and IVF embryos were also generated as controls, and all oocytes and embryos were analyzed using Monocle3 and DESeq2 to identify differentially regulated gene expression. We studied ZGA gene expression in late 2-cell stage embryos and identified clusters of samples with similar transcriptomes. Cluster 1 had no ZGA gene expression; Cluster 2 had early ZGA expression, including control early 2-cell stage IVF-derived embryos; Cluster 3 had moderate ZGA gene expression and contained the control late 2-cell stage IVF embryos that had high ZGA expression; and Cluster 4 had full ZGA expression. Surprisingly, all the 2-cell stage embryos developed from IVFC-derived oocytes initiated ZGA, while 54% of them represented “medium ZGA” and 46% showed “low ZGA.” This outcome indicates that all embryos analyzed from these IVFC-derived oocytes so far were ZGA-competent. Next, we looked at the expression of some marker genes, specifically, Mos (maternally deposited mRNA), Obox4 (zygotically expressed at early ZGA), and Duxbl (regulator of ZGA exit). All embryos were clear of the maternal Mos transcript, although embryos obtained from IVFC-derived oocytes showed slightly higher Mos gene expression. The early ZGA marker Obox4 was highly expressed in embryos developed from IVFC-derived oocytes compared with control embryos. Duxbl is the master regulator of ZGA exit, required for embryos to progress to the 4-cell stage. We found that Duxbl was expressed at lower levels in embryos developed from IVFC-derived oocytes, and current work aims to test if increasing Duxbl expression in these embryos can rescue their ZGA exit block and promote embryonic development. In conclusion, embryos developed from IVFC-derived oocytes can activate the ZGA program but have difficulty exiting this stage, likely leading to cleavage stage arrest.