Pharmacological blocking of h3k27 trimethylation alters the expression of polycomb repressive complex 2 genes in bovine blastocysts produced in vitro

Trimethylation of histone H3 on lysine 27 (H3K27me3) is established by Polycomb Repressive Complex 2 (PRC2) and it is associated with stable and heritable gene silencing. In pluripotent cells, genes associated with development and cell differentiation are maintained repressed by H3K27me3. However, this process is not fully understood. The AdoHcy hydrolase inhibitor 3-Deazaneplanocin A (DZNep) can block the action of the PRC2enzymes and thereby inhibit H3K27me3. In this study, we evaluated the effect of treating bovine embryos during in vitro development with DZNep on the expression of genes encoding PRC2 enzymes (EZH2, EED and SUZ12), and transcription factors regulating cell pluripotency (OCT4 and NANOG) and trophoblast differentiation (CDX2). Oocytes obtained from slaughterhouse ovaries were subjected to in vitro maturation (IVM) for 24 h at 38.5°C, with 5% CO2 in air and saturated humidity. In vitro fertilization (IVF) was performed with a previously tested frozen-thawed semen from a single Nellore bull. The oocytes and spermatozoa remained in coculture for 22 h under the same conditions of IVM. In D3 (considering the day of IVF as D0), the cleaved embryos were randomly allocated into four groups and exposed to 5 µM DZNep from: a) D3 to D5 (DZNep D3-D5); b) D3 to D8 (DZNep D3-D8); c) D5 to D8 (DZNep D5-D8); or d) without DZNep (Control Group). Embryos that developed to the blastocyst stage on D8 were collected for RNA extraction followed by qRT-PCR to assess abundance of transcripts. The experiment was repeated three times and all samples were analyzed in duplicate using 30 embryos per group. Total RNA was extracted using the PicoPure RNA isolation Kit (Life Technologies) and cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Life Technologies). Relative mRNA abundance was normalized to the levels of two reference genes (beta actin and 18S ribosomal). Data were analyzed using ANOVA and the means were compared by Dunnett’s test. DZNep treatment did not alter mRNA levels of SUZ12, NANOG, OCT4 and CDX2 in embryos that developed to the blastocyst stage. However, exposure to DZNep from day 3 to 8 increased mRNA levels of genes encoding the Polycomb enzymes EZH2 and EED. Findings from our previous studies confirmed that exposure of bovine embryos to DZNep during these periods of culture reduced blastocyst formation. These findings indicate that inhibition of H3K27me3 alters the regulation of Polycomb enzymes EZH2 and EED in early developing embryos, which suggests that these enzymes are involved in cell proliferation and blastocyst formation in the bovine embryo.