Is Biallelic Expression of the Imprinted Gene H19 in Preimplantation Mouse Embryos the Result of a Molecular Clock Rather than Embryo Manipulation?

Abstract

H19 is an imprinted non-coding RNA known to be transcribed from the maternally inherited allele. Genomic imprinting is defined as parent-specific gene expression. Previous research has shown maternal-specific expression of H19 in mouse blastocysts developed in vivo and biallelic expression in embryos cultured in vitro. In those studies embryos were collected at a particular stage of embryonic development (i.e. late blastocyst stage) despite the time disparity for embryos in different treatment groups to reach the desired developmental stage. For this study, we determined H19's allelic expression in embryos produced after ovarian hyperstimulation at specific times (84 hours, 96 hours, or 108 hours) following presumed ovulation, as opposed to specific stages of development. In addition, we determined the effects of two culture conditions (KSOM augmented with amino acids or Whitten's medium) on imprinted H19 expression in embryos cultured from the 2-cell stage, collected at 32 h post presumed ovulation, until 84, 96, or 108 h post-ovulation. RNA was collected and cDNA was synthesized from single embryos. Results showed that approximately 25% of embryos in all groups expressed biallelic H19 at 84 and 96 hours post-ovulation. Surprisingly, 50-77% of blastocysts expressed H19 from the normally silent paternal allele in all three treatment groups (i.e. in vivo produced, KSOM and Whitten's) at 108 hours post-ovulation. From these results we concluded that biallelic H19 expression is a normal event around the time of implantation in mouse embryos. However, recent data from our laboratory shows that superovulation can affect the level of global DNA methylation in growing oocytes. Therefore, we set out to determine if the high level of biallelic H19 expression was really the result of a developmental cue or simply an adverse outcome of the superovulation scheme. For the second study, we followed the same design as that described for embryos from superovulated females but this time the embryo donors did not receive hormonal stimulation. Briefly, females were observed for the presence of a copulatory plug to establish approximate time of ovulation. Embryos were either collected at the 2-cell stage and cultured as above or flushed from the uterus at 84 hours, 96 hours, or 108 hours. As in the previous experiment, cultured embryos were collected at equivalent times post ovulation. Again, RNA was collected and cDNA was synthesized from single embryos. Following PCR, products were restricted with an enzyme unable to cut the paternal allele allowing us to differentiate expression of each parental allele. Preliminary results show a similar pattern of biallelic expression in embryos produced after natural ovulation as those produced after superovulation. We suggest that during the peri-implantation stage, biallelic expression of H19 may be attributed to a molecular clock and not to be the result of embryo culture as has been previously concluded. (poster)