178 TRANSCRIPT ABUNDANCE OF METHYLTRANSFERASES SPECIFIC FOR H3K9 DIFFER AT DISCRETE STAGES OF PORCINE OOCYTE AND CLEAVAGE STAGE EMBRYO DEVELOPMENT

Abstract

Covalent modification of histone proteins plays a key role in transcriptional regulation. Methylation of the lysine residue 9 of histone protein 3 (H3K9) is globally remodeled during cleavage development. The aim of this study was to determine the relative transcript abundance of the 5 histone methyltransferases (HMTases) that have been shown to methylate H3K9, in porcine oocytes and cleavage stage embryos. We hypothesized that transcript levels for each of these HMTases (Suv39h1, Suv39h2, ESET, GLP, and G9a) would differ in their respective abundance at each developmental stage and that some would undergo dramatic changes in transcript abundance during development. To test this hypothesis, quantitative RT-PCR was performed on mRNA isolated from pools of 78–457 germinal vesicle (GV) and metaphase II (MII)-stage oocytes and 4-cell (4C) and blastocyst (BL)-stage embryos produced by either parthenogenetic activation (PA) or in vitro fertilization (IVF); each pool constituted an experimental replicate, and a minimum of 3 replicates were performed for each stage. Along with the 5 HMTases, transcripts for YWHAG were assayed and used as a normalizer. PCR data were quantified using Δ CT and 2^ΔΔ CT methods; in the latter case, Δ CT values from the GV stage were used as the calibrator. All data were analyzed by ANOVA and Tukey’s multiple-comparison test. In GV oocytes, Suv39h2 transcripts were in the highest abundance, while ESET transcripts were in the lowest abundance, ESET transcripts being 1450-fold less abundant than Suv39h2 (P < 0.05). In MII oocytes, Suv39h2, GLP, and G9a transcripts were present in highest abundance and were not significantly different from each other; Suv39h1 transcripts were 18-fold less abundant, and ESET transcripts were 6650-fold less abundant than GLP transcripts (P < 0.05). ESET transcripts were present in the least abundance in 4C-PA embryos, 84-fold less than GLP (P < 0.05); no difference in transcript levels for Suv39h1, Suv39h2, GLP, and G9a were detected. ESET transcripts were not detectable 4C-IVF embryos. ESET transcripts were present in the least abundance in BL-PA embryos, 39-fold less abundant than GLP; there was no difference in transcript abundance between Suv39h1, Suv39h2, and G9a. In BL-IVF-stage embryos, ESET transcripts were in the lowest abundance, 97-fold less abundant than GLP (P < 0.05). Analysis of how transcripts for each individual HMTase change from the GV oocyte to the BL embryo revealed no significant change in G9a transcript abundance. GLP transcripts decrease 7-fold at the 4C stage (GV v. 4C, P < 0.05). No difference in relative abundance of ESET transcripts was detectable at GV, MII, or BL-IVF stages; ESET was not detectable in 4C-IVF embryos. SuvH1 transcripts decreased 9-fold in BL-IVF (GV v. BL, P < 0.05); SuvH2 transcripts decreased 21-fold at the 4C-IVF and BL-IVF stages (GV v. 4C, BL, P < 0.05). The differences in transcript abundance of these genes indicate that their expression may change in order to regulate the transcription of genes important in early development.