198 Germline-Specific Expression of the Murine Oct4-EGFP Transgene in the Pig

Abstract

The Oct4 gene is crucial for undisturbed early embryonic development and maintenance of pluripotency in the mouse. It is found in mouse pre-implantation embryos after embryonic genome activation. After gastrulation, expression is restricted to germ cells. Limited research has been performed on OCT4 expression in the domestic pig, which is a valuable large animal model in biomedicine. Previously, we generated Oct4-EGFP reporter pigs carrying the genomic sequence of the murine Oct4 gene fused to the EGFP cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev. 20, 1563-1575, 10.1089/scd.2010.0399). In the present study, we used this animal model to analyse the expression profile of the murine Oct4-EGFP transgene in porcine oocytes, in vivo-derived embryos (4-cell embryos, 8- to 16-cell embryos, morulae, and blastocysts) and ovaries. We studied whether the murine Oct4-EGFP transgene mimics the expression pattern of the endogenous OCT4 protein in transgenic pigs. Immature oocytes were isolated from ovaries of Oct4-EGFP transgenic sows (n = 5) using slicing methods. For collection of porcine embryos, wild-type sows were inseminated with sperm from an Oct4-EGFP transgenic boar. Sows were sacrificed 3, 4, and 5 days after insemination, and embryos were recovered by flushing oviducts and uterus and analysed by confocal microscopy. Ovaries obtained from female animals (5–12 months) were enzymatically dissociated and analysed using flow cytometry. Immature oocytes (n = 19) showed a very low, diffuse EGFP signal in cytoplasm. Embryos up to the 4-cell stage (n = 45) did not show Oct4-EGFP transgene expression. For the first time, EGFP fluorescence was detected at the 8-cell stage (n = 29) and a strong EGFP signal was observed in 16-cell stages and morulae (n = 53). In blastocysts from Day 5 (n = 40) EGFP fluorescence was not restricted to the inner cell mass (ICM), but was also seen in the trophectoderm (TE). Expression of EGFP was not detected in ovarian cells (n = 12). Thereafter, we analysed the expression pattern of endogenous OCT4 protein by immunostaining in nontransgenic porcine oocytes and pre-implantation embryos. As in Oct4-EGFP transgenic embryos, no expression of OCT4 was observed in 4-cell embryos (n = 12). Nuclear staining first became visible at the 8-cell stage (n = 12), with a strong signal observed in 16-cell stages and morulae (n = 18). In blastocysts from Day 5 (n = 26), both ICM and TE cell nuclei showed expression of OCT4 protein. These results demonstrate that the Oct4-EGFP transgene expression pattern reproduces the endogenous OCT4 protein expression profile in porcine oocytes and pre-implantation embryos. The Oct4-EGFP transgene was first detected at the 8-cell stage, consistent with embryonic genome activation, which is initiated at the 4-cell stage. However, Oct4-EGFP expression was not detected in ovarian cells. This might be related to the very low expression pattern of the Oct4-EGFP transgene in primary oocytes. In summary, the Oct4-EGFP transgene in the pig provides a useful marker for monitoring pluripotency in pre-implantation embryos after embryonic genome activation. In ongoing experiments, we are analysing the expression profile of the Oct4-EGFP transgene and endogenous OCT4 protein in porcine pre-implantation embryos from Days 8 and 11.