The present study examined the activity and function of pig OCT4 (POU5F1) enhancer in porcine early embryo development. OCT4 is one of the master regulators for the pluripotency of early mammalian embryonic development and embryonic stem cells. It has two regulatory elements: distal enhancer (DE) and proximal enhancer (PE) in the upstream regulatory region. They are activated under different conditions such as naïve and primed. It is known that two enhancers are activated to produce OCT4 simultaneously or sequentially depending on the state of pluripotency. Many porcine OCT4 upstream region-based reporter systems have been reported because this is a necessary part of studying porcine-specific pluripotency. However, the porcine-specific OCT4 reporter system has never been transfected and functionally tested during porcine early embryo development. We performed functional tests of the previously established porcine-specific OCT4 reporter system in the early embryo development stage. Porcine embryos were micro-injected with the pOCT4-ΔPE-eGFP (DE-GFP) containing a distal enhancer and core promoter and pOCT4-ΔDE-DsRed2 (PE-RFP) containing a proximal enhancer and core promoter. They were cultured in PZM-3 at 39°C in a humidified atmosphere, 5% CO2, and 5% O2 for 168h. Fifty of the 100 embryos were injected with OCT4 reporter system as treatment groups and the other 50 were not injected (control groups). We analysed mRNA and protein expression of GFP and RFP using quantitative real-time PCR and confocal microscopy by developmental stages. The introduced reporter system could function beginning with the 4-cell stage. The expression of GFP and RFP was observed simultaneously in the early embryonic development stage up to blastocyst, indicating that porcine OCT4 was produced by distal and proximal enhancers, unlike mouse Oct4 expression, which was only controlled by the distal enhancer during early embryo development. Therefore, the mechanisms and functions of distal and proximal enhancers of porcine OCT4 were different from those of the mouse. This is similar to results of the previous experiment using porcine-induced pluripotent stem cells, which suggest that porcine OCT4 is expressed by two enhancers and in a stage-specific manner during reprogramming and that pigs do not use only one enhancer in pluripotent states, but two enhancers at the same time, and there is only a difference in degree. This work showed that the porcine OCT4 reporter system enables non-destructive analysis during early embryo development and it could be applied to study species-specific pluripotency and to help the establishment of naïve embryonic stem cells in the pig.
This work was supported by the BK21 Plus Program, the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1C1C1004514), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-3-HD020).
Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation
