Abstract
BackgroundThe lineage specification of mammalian embryos during preimplantation development has been studied for a long time but is still unclear. To understand the developmental process, many studies have examined lineage markers and mechanisms focusing on mouse embryos, but there are differences from human embryos. Pigs have been studied extensively in the field of disease model animals and xenotransplantation because of their physiological similarity with humans. Therefore, it is necessary to analyze gene expression patterns and lineage specification markers during early embryogenesis in pigs, a model animal similar to humans.ResultsAnalysis of the expression pattern of the core pluripotent factors (OCT4, SOX2 and NANOG) of preimplantation porcine embryos showed that SOX2 was only expressed in some cells from the early stage, so SOX2 was selected as an ICM inducible factor candidate. Next, transcript and protein expression patterns were estimated at the early stage (Day 5) and late stage (Day 7) of blastocysts injected with the CRISPR Cas9 system selected through gRNA validation. An ICC assay revealed that the expression of ICM-related genes (SOX2, NANOG and SOX17), except OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes (NANOG, SOX17 and SMAD7), excluding OCT4, and proliferation-related genes (KDM8 and DDB1) were decreased in SOX2-targeted blastocysts, which showed more differences in late-stage blastocyst than in early-stage blastocyst. Last, in SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased, but the ICM/TE ratio decreased.ConclusionsTaken together, our results demonstrated SOX2 is essential for ICM formation and cell proliferation in porcine early stage embryogenesis. These findings will help to elucidate gene regulation related to lineage specification during porcine early development.
Anoctamin 1 Positive Esophageal Interstitial Cajal Cells in Late Stage Human Embryos