100 GLOBAL H3K27me3 IS DISTINCT IN THE PORCINE EPIBLAST AND TROPHECTODERM AND IS POTENTIALLY CORRELATED TO X-INACTIVATION IN FEMALE EMBRYOS

Abstract

During embryonic development in mammals, the first cell fate decision occurs at the morula stage, which leads to the establishment of the inner cell mass (ICM) and trophectoderm (TE). At this point of development, differential patterns of gene expression and epigenetic marks are observed within these two lineages. The ICM later differentiates to form the epiblast. Previous reports suggest that the distinct patterns of expression might be substantially regulated by epigenetic marks such as DNA methylation and post-transcriptional modifications. In this study, we examined the global H3K27me3 distribution, as well as the expression levels of H3K27me3 specific methyltransferases and XIST in the epiblast and TE of Day 10 in vivo porcine embryos. A total of 33 embryos were collected by non-surgical flushing from inseminated, and later, culled sows. The embryos were sexed by PCR (sequences of ZFX (X chromosome) and SRY (Y chromosome) genes were amplified by PCR using primers) because the H3K27me3 has been associated with X chromosome inactivation. For immunocytochemistry, a small piece of TE was removed before fixation and analysed for sexing by PCR. For comparative RT-PCR studies, embryos were mechanically separated, sexed, and then later pooled as male or female epiblast and TE (male = 6, female = 8). Global H3K27me3 was analysed by immunocytochemistry in 11 male and 8 female Day 10 embryos. Expression of methylases (EZH2, EED and SUZ12, three core components of PRC2), demethylases (JMJD3 and UTX) of H3K27me3, and XIST was performed on the pooled epiblasts and pooled TE. Expression levels were normalized to the reference gene, GAPDH, and was further normalized to Day 9 embryos. Our results show that high nuclear expression of H3K27me3 was observed in both male and female TE cells, with little to no observable expression in the epiblast. However, a single, small, punctate spot could be detected within the nuclei of the female epiblast and TE. XIST, a non-coding RNA associated with the initiation of X chromosome inactivation (XCI), was observed to be highly expressed in the female epiblast and TE, which suggests H3K27me3 punctate spots that presented in female epiblast or TE are potentially expressed on the inactive X chromosomes. We also detected higher expression of the H3K27me3 methylase (EZH2) and the methylase cofactors (EED, SUZ12) in both male and female TE. Of interest, EED expression was higher in the female epiblast and TE compared to the male epiblast and TE. This suggests that EED may play an important role in the initiation of XCI. The expression of H3K27me3 demethylases JMJD3 and UTX, were also higher in the TE compared to the epiblast, which indicates the trimethylation of H3K27 in the embryos is a dynamic process. We suggest that no, or extremely low, H3K27me3 in the porcine epiblast might be required for the cells to program gene expression towards different cell fates upon differentiation and the enrichment of H3K27me3 in the TE of Day 10 porcine embryos might reinforce the commitment towards the TE lineage.