44 Analysis of abnormal chromatin configuration induced by inhibiting MEK at the 1-cell stage

After fertilisation, mammalian oocytes resume meiosis, leading to pronuclear (PN) formation. It has been known that the mitogen-activated protein kinase pathway negatively regulates PN formation. However, it remains largely unknown how the mitogen-activated protein kinase pathway is involved in the dynamic organisation of chromatin structure in PNs. Here, to address how MEK1/2 activity is involved in PN formation and transcriptional regulation, we examined whether treatment of fertilised oocytes with MEK1/2 inhibitor (PD0325901; PD) during the 1-cell stage affects their global epigenetic modifications and heterochromatin formation in mice. We performed IVF using superovulated oocytes from and ICR strain female mouse and treated IVF embryos with 1 µM PD for 10h from insemination to observe global epigenetic modifications such as H3K9me3 or H3K27me3, localization of heterochromatin protein. First, we found that PD treatment enhanced PN formation with abnormal pronuclear sizes, that is, larger female and smaller male PNs. Further, PD treatment for 10h after fertilisation caused significantly lower global H3K9me3 or H3K27me3 modifications in female PN [H3K9me3: 1.00±0.01 vs. 0.75±0.01 (N>10); P=1.0×10−2, H3K27me3: 1.00±0.01 vs. 0.55±0.01 (N>10); P=6.0×10−7], suggesting failure of establishment of normal histone modifications for heterochromatin. Consistently, we found loss of HP1b around the nucleolus precursor body, which is normally associated with pericentric heterochromatin, suggesting that MEK1/2 activity is required for normal PN and heterochromatin formation. Regardless, embryos treated with PD at the 1-cell stage recovered such global histone modifications through the pre-implantation embryos to reach the blastocyst stage. Together, our data show that MEK1/2 activity plays a pivotal role not only in regulation of PN formation but also heterochromatin formation during the 1-cell stage in mice.