SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre‐implantation embryo development

• Published in: Cell proliferation 2021-08, Vol.54 (8), p.e13097-n/a
• Main Authors: Lee, Mingyun, Choi, Kwang‐Hwan, Oh, Jong‐Nam, Kim, Seung‐Hun, Lee, Dong‐Kyung, Choe, Gyung Cheol, Jeong, Jinsol, Lee, Chang‐Kyu
• Format: Article
• Language: English
• Subjects: Animals; Blastocyst - cytology; Blastocyst - metabolism; Blastocysts; Cell Lineage; Cell number; Cell Proliferation; Cloning; Comparative studies; CRISPR; CRISPR-Cas Systems - genetics; CRISPR/Cas9; embryo; Embryo, Mammalian - cytology; Embryo, Mammalian - metabolism; Embryogenesis; Embryonic Development; Embryonic growth stage; Embryos; Female; Gene expression; Gene regulation; Genes; Genetic engineering; Genetic testing; Immunocytochemistry; Implantation; Kinases; Laboratory animals; Mammals; Oct-4 protein; Original; pig; Pluripotency; RNA, Guide, CRISPR-Cas Systems - metabolism; SOX2; SOXB1 Transcription Factors - genetics; SOXB1 Transcription Factors - metabolism; Stem cells; Swine; Transcription factors; Transcription Factors - genetics; Transcription Factors - metabolism; Transcriptome
• ISSN: 0960-7722; 1365-2184
• DOI: 10.1111/cpr.13097

Objectives Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre‐implantation embryo development. The extraordinarily longer pre‐implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre‐implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre‐implantation embryos between species. Materials and methods To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss‐ and gain‐of‐function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real‐time PCR. Results Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2‐disrupted blastocysts, the expression of the ICM‐related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real‐time PCR analysis, pluripotency‐related genes, excluding OCT4, and proliferation‐related genes were decreased in SOX2‐targeted blastocysts. In SOX2‐overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased. Conclusions Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early‐stage embryogenesis. We analysed the phenotypes of SOX2 knockout and SOX2‐overexpressing pre‐implantation porcine embryos to understand the function of SOX2, one of the core transcription factors. In SOX2‐targeted embryos, total cell number decreased, and expression of pluripotent‐related genes and proliferation‐related genes decreased. Loss‐ and gain‐of‐function analysis revealed that SOX2 regulates total cell number and expression of pluripotent‐related genes and proliferation‐related genes. These results indicate that SOX2 plays important role in cell proliferation and ICM formation in porcine pre‐implantation embryos.