144 EPIGENETIC ASYMMETRY IN HISTONE H3 LYSINE 9 DIMETHYLATION STATUS IN PRONUCLEAR STAGE PORCINE EMBRYOS

Abstract

Covalent modification of specific residues on the tail regions of core histone proteins has been shown to play a key role in regulation of the genome. Methylation of lysine 9 on histone H3 (H3K9) is associated with repression of transcription and formation of heterochromatin domains. The maternal- and paternal-derived pronuclei from pronuclear stage murine embryos have an asymmetric distribution of H3K9 dimethylation between the 2 pronuclei; maternal pronuclei have the H3K9 dimethylation, whereas the paternal pronuclei lack this modification. The aim of this study was to characterize the H3K9 dimethylation pattern in cleavage stage porcine embryos. Indirect immunocytochemical staining was performed using a commercially available antibody from Upstate (Charlottesville, VA) that recognizes the dimethylated form of lysine 9 on histone H3 and fluorescein isothiocyanate (FITC)-conjugated secondary antibody on germinal vesicle (GV) stage porcine oocytes and cleavage stage porcine embryos. Germinal vesicle stage oocytes were matured in vitro for 44 h in a chemically defined maturation medium (TCM 199 supplemented with 0.1% PVA, 0.069 mg/mL cysteine, 10 ng/mL EGF 0.5 IU/mL LH, and 0.5 IU/mL FSH) at 39°C in 5% CO₂. Following in vitro fertilization, presumptive zygotes were cultured in NCSU23 medium containing 4 mg/mL BSA at 39°C in 5% CO₂, 5% O₂. Embryos were fixed in 3.7% paraformaldehide for 2 h and washed in PBS and 0.1% Tween20. H3K9 dimethylation is present in the nuclei of GV-stage oocytes (n = 24) and pronuclear (n = 13), 2-cell (n = 4), 4-cell (n = 9), blastocyst (n = 8) stage embryos. The analysis revealed 2 interesting findings. First, examination of thin optical sections through the nuclei of processed embryos on a confocal microscope revealed that in GV-stage oocytes and pronuclear, 2-cell and 4-cell stage embryos the dimethylated form of H3K9 was distributed throughout the nuclei at these developmental time points, whereas in blastocyst stage embryos, the dimethylated H3K9 was restricted to the nuclear periphery. Second, not all pronuclei within pronuclear stage embryos were positive for the dimethylated form of H3K9. To determine if differential H3K9 methylation pattern observed among pronuclei in 1-cell stage embryos was due to parental origin, we produced parthenogenic porcine embryos by electrically activating porcine oocytes in calcium containing activation medium and processed them as indicated above for fertilized oocytes (n = 13). All pronuclei found in pronuclear stage parthenogenic porcine embryos were positive for dimethylation of H3K9. Chi square analysis revealed this pattern to be different from that observed in pronuclear embryos produced by fertilization (P < 0.05). In summary we conclude that the pronuclei in 1-celled embryos produced by in vitro fertilization are differentially dimethylated at H3K9 and that the localization of dimethylated H3K9 changes during cleavage development.