177 EXPRESSION PROFILING OF HISTONE-MODIFYING GENES IN BOVINE PREIMPLANTATION EMBRYOS

Abstract

Global activation of the embryonic genome marks one of the most important aspects of early embryonic development and coincides with both global DNA and histone modifications necessary to control gene expression for further development. In bovine preimplantation embryos, epigenetic reprogramming occurs between fertilization and the blastocyst stage. De novo DNA methylation from the 8-cell stage to the morula represents activation of DNA methyltransferases and histone-modifying genes; however, it is still unclear how these critical events of early development and differentiation are regulated. In this preliminary study, we used quantitative real-time PCR (qRT-PCR) to examine stage-specific expression in bovine gametes (testis and ova) and preimplantation embryos of eight histone-modifying genes: LSH1 (which associates with both DNA and histone methyltransferases), LSD1 (a histone demethylase), SUV39H1, SUV420H1, SETB1, SUZ12, SMYD3, and G9a (all representative histone methyltransferases). Bovine ova and embryos were produced via in vitro maturation, fertilization, and culture from a single pool of ova as per standard laboratory protocol. Testes were harvested from a mature bull and immediately snap frozen in liquid nitrogen. RNA was isolated from testis, and groups of 10–15 in vitro-matured bovine ova or in vitro-produced embryos at the 2-, 8-cell, morula, and blastocyst stages and stored at –80°C until further use. cDNA was generated from each RNA sample using Superscript II" reverse transcriptase (Invitrogen, Carlsbad, CA, USA) under identical PCR conditions. Relative gene expression from each RNA sample was calculated in triplicate using the ΔΔCt method with both SYBR^® Green and Taqman^® qRT-PCR methods (Applied Biosystems, Foster City, CA, USA) and normalized to endogenous bovine GAPDH expression as per standard protocol. Due to excessive variation in a few of the qRT-PCR reactions, consistent data were collected for only SETB1, SUV39H1, SUV420H1, and G9a. SETB1 and G9a shared a similar expression pattern, with both exhibiting a dramatic relative increase in transcript level near the 8-cell stage and reduction to basal levels thereafter. Also, SUV39H1 and SUV420H1 were similar, showing a gradual increase in relative expression from the oocyte to the 8-cell stage, followed by a drop to low levels by the morula stage. These data indicate that several histone-modifying genes are expressed in distinct patterns during the critical period of zygotic genome activation and establishment of the epigenome in early preimplantation bovine embryos. Continuing studies will determine specific gene function through shRNA-induced silencing of histone-modifying genes and their role in control of gene expression.