Maternal Vs. Embryo Genes: Which One Is More Important For Embryo Culture?

Abstract

The timing of human genome activation and maternal transcript degradation in preimplantation stage embryos has never been fully investigated. As a result the relative importance of maternal genes and embryonic genes in the control of early development is unclear. In mice, maternal mRNAs are largely down-regulated at the 2-cell stage, accompanied by a surge of newly transcribed message from embryonic genome activation (EGA). In human, although it is speculated that these events occur at a later stage of embryo development, the exact timing/mechanism has not been established. The objective of this study was to elucidate the relative importance of maternal and embryonic genes in preimplantation development by determining the timing of maternal transcript degradation and EGA by using microarray technology. Laboratory study A total number of 201 embryos at various stages, including zygote, 2-, 4-, 8-cell, morulae, and blastocysts were collected into individual collection tubes for RNA extraction in each of three replicates. PicoPure RNA Isolation Kit (Arcturus) was used and amplification was performed by two-round in vitro transcription with Ribo-Amp RNA Amplification Kit (Arcturus). Biotin-labeled amplified probes were hybridized to Affymetrix microarray (HG-U133 plus 2.0 Array). Scanned arrays were analyzed using dChip software. Of more than 10,000 genes analyzed, only 3 and 4 genes were down-regulated at zygote to 2, and 2-cell to 4-cell transition, respectively. However, 3,462 genes were down-regulated when the embryos developed from the 4 to 8-cell stage. This finding strongly suggests that maternal transcript degradation in human preimplantation embryos takes place between the 4 and 8-cell stage. Several known maternal transcripts, such as zona pellucida glycoprotein 2 and 4, showed expression patterns of rapid degradation between 4-/8-cell stage in our data, corresponding to our global analysis mentioned above. This finding in human embryos is significantly different from our previous mouse study, which showed that maternal messages degrade when embryos develop from the zygote to 2-cell stage. Very few new transcripts were observed before the 4-cell stage (1 in zygote/2-cell, 30 in 2/4-cell stage, respectively). Between 4/8-cell stage, a surge of new transcripts (756 genes) was observed, followed by 632 genes in 8-cell/morulae and 877 genes in morulae/blastocyst stage. This indicates that EGA is concomitant with maternal message degradation, when embryos develop from the 4 to 8-cell stage. The characteristics of deposited maternal transcripts were also determined. In human embryos, these maternally deposited RNAs are enriched with alternative polyadenylation sites with a ratio of 3.74 (p <0.05), where 36.3% genes are polyadenylated while 9.7% are not. With the expression of more than 10,000 genes analyzed by microarray technology, we found maternal that mRNA degradation and embryo genome activation both first occur when embryos develop from 4 to 8-cell. This strongly suggests that maternal genes play a more important role in the first two days of embryo development than previously expected. Maternal genes may be mainly responsible for early embryo development before genome activation, when newly transcribed embryo genes begin to take over. The impact of maternal message on preimplantation embryo culture, cell cycle, and apoptosis is currently under investigation.