82 Stage-specific expression of lineage marker genes and pluripotency marker distribution in porcine pre-implantation embryos

Abstract

Lineage specification in pre-implantation embryos has been revealed, and it was expedited recently by single cell studies. However, data on expression marker genes and proteins in porcine embryos were still missing. We aimed to investigate the expression and distribution of marker genes and proteins, respectively, in IVF and parthenogenetically activated (PA) embryos. For this, cumulus-free oocytes were co-incubated with sperm in modified Tris-buffered medium (mTBM) for 5h and PA was performed using an electric pulse in activation medium. Following this, the embryos were incubated in porcine zygote medium 3 (PZM3). We first tested gene expression level of lineage candidates (internal control; β-actin). In IVF embryos (30, 25, 20, 15, 10, and 5 embryos pooled on Day 2, 3, 4, 5, 6, and 7; replicated 3 times), trophectoderm (TE)-specific genes (Dab2, Gata3) showed peaks on Day 4-5. Within the 2 genes, Dab2 had an earlier peak than Gata3. Inner cell mass (ICM) marker candidates (Nanog, Sox2, and Hnf4a) had diverse patterns. The Nanog and Sox2 genes had peak expression on Day 3. The Nanog expression dropped gradually, but Sox2 dropped suddenly on Day 4. Otherwise, Hnf4a expressed little in Day 3 and expression was sustained from Day 4 to 7. Primitive endoderm markers showed the highest expression on Day 4. We also checked expression level of ICM markers (Sox2, Oct4, and Nanog) in PA embryos (20, 20, 20, 10, and 5 embryos were pooled in 2, 4, 6-8 cells, morula, early, and late blastocyst stages; replicated 3 times). Expression of markers was similar (the highest in the 6-8-cell stage; at least 7.3-, 4.5-, and 3.7-fold compared with the other stages in Sox2, Oct4, and Nanog). We used analysis of variance and Tukey's test for statistical analysis. Following this, we conducted immunocytochemistry with both IVF and PA embryos (20 in each condition). Primary antibodies were treated overnight at 4°C and appropriate secondary antibodies were treated 1h at room temperature. In the case of IVF, well-known ICM markers (SOX2, OCT4, NANOG, and SOX17) showed restricted distribution in nuclei of ICM cells. However, DAB2 was distributed in the cytoplasm of TE cells. In PA embryos, SOX2 and NANOG distributions were similar to IVF. The OCT4 in ICM cells from morula to early blastocyst was restricted, but not in Day 7 embryos. In conclusion, marker genes showed diverse expression pattern in IVF, but all ICM-specific genes had a similar pattern in PA. Also, ICM marker proteins were restricted in nuclei of ICM cells only except Day 7 PA. Our results provide eye-opening information on marker contribution to lineage specification of porcine embryos. This work was supported by the National Research Foundation in Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03032256, NRF-2019R1C1C1004514).