Determination of Allelic Expression of H19 in Peri-Implantation Mouse Embryos.

Abstract

H19 is a maternally-expressed imprinted non-coding RNA with tumor suppressor activity. During mouse preimplantation development, H19 is primarily expressed in the trophectoderm cells. It has been documented that preimplantation embryo manipulation affects the allelic expression of this gene causing it to become biallelically expressed. We have observed biallelic expression of H19 in embryos that developed in vivo leading us to hypothesize that H19 biallelic expression might be a normal occurrence in mouse embryos immediately prior to implantation. Expression of H19 was determined in C7xB6 F1 embryos derived from superovulated C57BL/6J(Cast7) (C7) females mated to C57BL/6J (B6) males. In vivo-produced control embryos were collected from the uterus. In vitro-cultured embryos were collected from the oviduct at the 2-cell stage and cultured in KSOM supplemented with amino acids or Whitten's media. The embryo collection times chosen were 84, 96, and 108 hours post presumed ovulation to obtain embryos at the early, mid, and late blastocyst stages of development. QPCR with subsequent FRET or RFLP followed by PAGE allowed us to determine allele-specific gene expression in single embryos. For an embryo to be considered to have biallelic H19 expression, =10% of the total H19 RNA must be paternal in origin. So far we have analyzed H19 expression in 21, 32, 47 in vivo produced embryos (for 84, 96, 108 h; respectively), 18, 52, 58 KSOM cultured embryos, and 11, 44, 31 Whitten's cultured embryos. Those results show that the paternal allele is expressed in 24,31, 77 % of in vivo produced embryos (for 84, 96, 108 h respectively), 28, 31, 60 % of KSOM cultured embryos and 18, 15, 55 % of Whitten's cultured embryos. As recent reports suggest a possible effect of superovulation on imprinting, we are repeating the experiment with embryos produced after natural ovulation. Thus far we have analyzed H19 expression in 5, 25, 15 in vivo produced embryos (for 84, 96, 108 h, respectively), 1, 19, 25 KSOM cultured embryos and 0, 8, 20 Whitten's cultured embryos. Those results show that the paternal allele is expressed in 20, 32, 80 % of in vivo produced embryos (for 84, 96, 108 h respectively), 0, 32, 60 % of KSOM culture embryos and not detected, 25, 30 % of Whitten's cultured embryos. We are currently performing DNA methylation assays to ascertain if the observed loss of imprinting correlates with a concomitant decrease in DNA methylation at the H19 regulatory region. Peri-implantation mouse embryos are composed of at least five cell types, namely, inner cell mass, primitive endoderm, mural trophectoderm, polar trophectoderm, and trophoblast giant cells. We speculate that biallelic H19 expression is the result of the differentiation of the trophectoderm trophoblast giant cells and that this may be relevant for implantation. We are currently performing microdissection studies to divide the embryo in two-three parts, one containing the inner cell mass cells, primitive endoderm cells and/or adjacent polar trophectoderm and the other containing the mural trophectoderm and primary trophoblast giant cells. Using a combination of TaqMan probes for H19, Igf2, Pou5f1, Cdx2, Rdx, Dek we intend to ascertain the enrichment of giant cells in our mural trophectoderm sample. Initial work in this area demonstrates our ability to have mural trophectoderm cells free or with minimal inner cell mass contamination based on lack of expression of Pou5f1 in this embryo section.