Changes in OCT4 expression play a crucial role in the lineage specification and proliferation of preimplantation porcine blastocysts

Abstract

Objectives: Curiosity about the role of OCT4, a core transcription factor that maintains inner cell mass (ICM) formation during preimplantation embryogenesis and the pluripotent state in embryonic development, has long been an issue. OCT4 has a speciesspecific expression pattern in mammalian preimplantation embryogenesis and is known to play an essential role in ICM formation. However, there is a need to study new roles for OCT4-related pluripotency networks and second-cell fate decisions. Materials and Methods:To determine the functions of OCT4 in lineage specification and embryo proliferation, loss-and gain-of-function studies were performed on porcine parthenotes using microinjection. Then, we performed immunocytochemistry and quantitative real-time polymerase chain reaction (PCR) to examine the association of OCT4 with other lineage markers and its effect on downstream genes.Results: In OCT4-targeted late blastocysts, SOX2, NANOG, and SOX17 positive cells were decreased, and the total cell number of blastocysts was also decreased. According to real-time PCR analysis, NANOG, SOX17, and CDK4 were decreased in OCT4-targeted blastocysts, but trophoblast-related genes were increased. In OCT4-overexpressing blastocysts, SOX2 and NANOG positive cells increased, while SOX17 positive cells decreased, and while total cell number of blastocysts increased. As a result of real-time PCR analysis, the expression of SOX2, NANOG, and CDK4 was increased, but the expression of SOX17 was decreased. Conclusion:Taken together, our results demonstrated that OCT4 leads pluripotency in porcine blastocysts and also plays an important role in ICM formation, secondary cell fate decision, and cell proliferation. | INTRODUCTIONMammalian embryos undergo two cell fate decisions during preimplantation embryogenesis. 1 The first decision is divided into an inner cell mass and trophectoderm, 2 and the second decision segregates ICM into epiblast and primitive endoderm. 3,4 Numerous genes and mechanisms are involved in this lineage specification, and OCT4 has been heavily studied due to it's importance. OCT4 is one of the core transcription factors of pluripotency, has a DNAbinding domain, and controls expression by binding to a consensus

Maslinic Acid Supplementation during the In Vitro Culture Period Ameliorates Early Embryonic Development of Porcine Embryos by Regulating Oxidative Stress

“…The downregulation of the proapoptotic gene BAX and upregulation of the antiapoptotic gene BCL2 in the MA group also supported our hypothesis [ 42 ]. In addition, OCT4 plays a vital role in both cell fate decisions and cell proliferation in porcine early-stage embryos [ 43 ], while knockout of OCT4 inhibits blastocyst development [ 44 ]. Therefore, the OCT4 expression level is positively related to blastocyst pluripotency.…”

Improving the developmental competences of porcine parthenogenetic embryos by Notoginsenoside R1‐induced enhancement of mitochondrial activity and alleviation of proapoptotic events

“…Functionally, Nanog works in concert with OCT4 . These genes are integral for maintaining embryonic stem cell pluripotency and promoting cell proliferation, and represent promising constituents for improving embryonic developmental quality (Hisey et al, 2021; Lee et al, 2022; Pan & Thomson, 2007) since IVC culture tends to accumulate excessive ROS levels that inhibit embryonic development (Kordowitzki, 2021; Wang, Tang, et al, 2021; Wang, Wu, et al, 2021). Therefore, after establishing NGR1 as effective in providing early development in porcine embryos, this study demonstrated that antioxidant NGR1 supplementation compensated for the IVC culture deficiency by increasing GSH and NRF2 levels while decreasing ROS.…”

Electroporation‐mediated genome editing in vitrified/warmed porcine zygotes obtained in vitro

“…In the present study, however, OCT4 ‐GE blastocysts, although produced in vitro, did not show expansion or hatching after one additional day of culture until Day 7. Lee et al (2022) reported that OCT4 knockout in parthenogenetic porcine embryos decreased total cell number in blastocysts. Our results using fertilized oocytes are consistent in this regard.…”

“…In mice and pigs, OCT4 expression does not seem to be a prerequisite for blastulation (Lee et al, 2022; Nichols et al, 1998). On the other hand, to our knowledge, the importance of CD46 expression for in vitro development of porcine embryos has not been investigated to date.…”

“…As a knock‐out model, we targeted the OCT4 (NM_001113060.1, also known as POU5F1 ) and CD46 (NM_213888.1, also known as MCP ) genes because we already knew from our previous studies (unpublished) that these genes are expressed at the blastocyst stage. In addition, OCT4 is not required for blastocyst formation in mice (Nichols et al, 1998) and pigs (Lee et al, 2022). Therefore, it was expected that the efficacy of GE would be easily verified by sequencing the target DNA of the resulting blastocysts as well as by immunocytochemical analysis.…”