Enhancer of zeste homolog 2 (EZH2), a component of the PRC2 complex, trimethylates H3K27, a transcriptionally repressive histone mark. EZH2 is encoded by a dormant maternal mRNA and inhibiting the maturation-associated increase in EZH2 activity using either a combined siRNA/morpholino approach or a small molecule inhibitor (GSK343) inhibits development of diploidized parthenotes to the blastocyst stage but not inseminated eggs, with longer GSK343 treatments leading to progressively greater inhibition of development. GSK343 treatment also results in a decrease in H3K27me3 and a decrease in global transcription in 2-cell parthenotes but not 2-cell embryos derived from inseminated eggs. RNA-sequencing revealed the relative abundance of ~100 zygotically-expressed transcripts is decreased by GSK treatment in parthenotes, but not in embryos, with many of the affected transcripts encoding proteins involved in transcription. A previous study found that parthenotes deficient in maternal Ezh2 readily develop to the blastocyst stage. To reconcile these differences we propose that the H3K27me3 state present in the zygote needs to be faithfully propagated following DNA replication in at least one pronucleus, otherwise development is compromised.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression after transcription. miRNAs are present in transcriptionally quiescent full-grown oocytes and preimplantation embryos that display a low level of transcription prior to embryonic genome activation. The role of miRNAs, if any, in preimplantation development is not known. The temporal pattern of expression of miRNAs during bovine preimplantation development was determined by small RNA-sequencing using eggs and preimplantation embryos (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst). Embryos cultured in the presence of α-amanitin, which permitted the distinguishing of maternal miRNAs from embryonic miRNAs, indicated that embryonic miRNA expression was first detected at the two-cell stage but dramatically increased during the morula and blastocyst stages. Targeting DGCR8 by a small-interfering RNA/morpholino approach revealed a role for miRNAs in the morula-to-blastocyst transition. Knockdown of DGCR8 not only inhibited expression of embryonically expressed miRNAs but also inhibited the morula-to-blastocyst transition. In addition, RNA-sequencing identified an increased relative abundance of messenger RNAs potentially targeted by embryonic miRNAs in DGCR8-knockdown embryos when compared with controls. Results from these experiments implicate an essential role for miRNAs in bovine preimplantation embryo development.

Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development.