Document Type : Original Article
Department of Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran ;Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
4National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
5Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran
6Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Chimeric animal exhibits less viability and more fetal and placental abnormalities than normal animal. This study was aimed to determine the impact of mouse embryonic stem cells (mESCs) injection into the mouse embryos on H3K9me3 and H3K4me3 and cell lineage gene expressions in chimeric blastocysts.
Materials and Methods
In our experiment, at the first step, incorporation of the GFP positive mESCs (GFP-mESCs) 129/Sv into the inner cell mass (ICM) of pre-compacted and compacted morula stage embryos was compared. At the second and third steps, H3K4me3 and H3K9me3 status as well as the expression of Oct4, Nanog, Tead4, and Cdx2 genes were determined in the following groups: i. In vitro blastocyst derived from in vivo morula subjected to mESCs injection (blast/chimeric), ii. In vivo derived blastocyst (blast/in vivo), iii. In vitro blastocyst derived from culture of morula in vivo (blast/morula), and iv. In vitro blastocyst derived from morula in vivo subjected to sham injection (blast/sham).
Subzonal injection of GFP-mESCs at the pre-compacted embryos produced more chimeric blastocysts than compacted embryos (P < 0.05). The number of trophectoderm (TE), ICM, ICM/TE and total cells in chimeric blastocysts were less than the corresponding numbers in blastocysts derived from other groups (P < 0.05). In ICM and TE of chimeric blastocysts, the levels of H3K4me3 and H3K9me3 were respectively decreased and increased compared to the blastocysts of the other groups (P < 0.05). Expressions of Oct4, Nanog and Tead4 were decreased in chimeric blastocysts compared to the blastocysts of the other groups (P < 0.05), while this was not observed for Cdx2.
In the present study, embryo compaction significantly reduced the rate of incorporation of injected mESCs into the ICM. Moreover, in chimeric blastocysts, the levels of H3K9me3 and H3K4me3 were altered. In addition, the expressions of pluripotency and cell fate genes were decreased compared to blastocysts of the other groups.