Running title: H19 expression in peri-implantation mouse embryos

H19 is a maternally-expressed imprinted non-coding RNA with tumor suppressor activity. During mouse pre-implantation development, H19 is primarily expressed in the trophectoderm cells. The purpose of this project was to determine allelic expression of H19 in pre- and peri-implantation mouse embryos. We were further interested in determining if loss of imprinted H19 expression during blastocyst development occurred as a result of superovulation and/or culture. Our last goal was to ascertain if differential H19 allelic expression occurred between the inner cell mass (ICM)containing half and the primary trophoblast giant cell (PTGC)-containing half of the embryo. C57BL/6J (Cast-7) xC57BL/6J F1 embryos were collected from the uterus at 84, 96, and 108 h following natural ovulation or superovulation. In vitro-cultured F1 embryos were harvested from the oviduct at the 2-cell stage and cultured in KSOM + aa supplemented with amino acids or Whitten media and collected at the above-mentioned times. Allele-specific H19 expression in single embryos was determined by qRT-PCR followed by fluorescence resonance electron transfer or RTPCR followed by restriction fragment length polymorphism and polyacrylamide gel electrophoresis (RFLP-PAGE). Peri-implantation embryos were microdissected into two sections, one containing the ICM and the other containing the PTGC. TaqMan probes for Dek, Pou5f1, Itga7, H19 and Igf2 were used to ascertain gene expression enrichment in each section. Allele-specific H19 expression in embryo sections was determined by RFLP-PAGE. We conclude that loss-of-imprinting of H19 occurs in the PTGC-containing section of peri-implantation mouse embryos. We speculate that this is part of a physiologic event at the time of implantation in the mouse. Summary sentence: Mouse embryos normally express H19 from both parental-alleles in the primary trophoblast giant cell-containing section at peri-implantation.