51 CHARACTERIZATION OF HISTONE H3 LYSINE 27 TRI-METHYLATION AND POLYCOMB GROUP GENE EXPRESSION IN BOVINE FERTILIZED, PARTHENOGENETIC, AND CLONED EMBRYOS

Abstract

The low developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos has been attributed to aberrant or incomplete epigenetic reprogramming. Tri-methylation of Histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes (Ezh2, Eed, and Suz12) and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes during bovine preimplantation development and to compare the dynamics of H3K27me3 after IVF, parthenogenetic activation (PG), and SCNT. Embryos were produced using our standard protocols (Ross et al. 2006 Biotechniques 41, 741–750). MII oocytes and pronuclear-, 2-cell-, 4-cell-, 8-cell-, morula-, and blastocyst-stage embryos were collected for quantitative RT-PCR or immunofluorescence (IF). IF analysis of embryos was performed using primary antibodies against H3K27me3, Eed, Ezh2 (Abcam, Cambridge, MA, USA), and Suz12 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and imaged by confocal microscopy. The average background-corrected nuclear fluorescence intensity for each embryo was analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Ezh2, Eed, Suz12, and control transcript levels from oocytes and IVF-derived embryos were determined by absolute quantification as described by Bettegowda et al. (2006 Mol. Reprod. Dev. 73, 267–278). RNA levels were normalized to the external control and analyzed by ANOVA (mixed model). A steady decrease in total transcripts was observed from the MII to the 8-cell stage (P < 0.05). During this period, the levels of polyadenylated Suz12 and Eed transcripts remained steady, while Ezh2 transcripts increased at the 2-cell stage (P < 0.05) and then decreased from the 2- to 4-cell stage (P = 0.10). After embryonic genome activation, all three genes displayed an increase in transcript abundance by the blastocyst stage (P < 0.05). Concurrent IF analysis revealed that Ezh2 localized to the nucleus throughout preimplantation development, Eed localized to the cytoplasm at the 2-, 4- and 8-cell stages (expression confirmed by westernblot) and to the nucleus at morula and blastocyst stages, and Suz12 was exclusively nuclear and detected only at morula and blastocyst stages. IF analysis of H3K27me3 revealed that, for all embryo types, nuclear fluorescent intensity was highest at the MII stage, steadily decreased to reach a nadir at the 8-cell stage (P < 0.05), and then significantly increased at the blastocyst stage (P < 0.05). H3K27me3 fluorescent intensity was significantly higher in SCNT blastocysts than in IVF and PG blastocysts (P < 0.05). We conclude that H3K27me3 is efficiently removed from the embryonic and somatic cell chromatin, but is abnormally reestablished in SCNT embryos.