Expression Patterns Of GCN5 And HDAC1 In Preimplantational Mouse Embryos And Effects Of In-vitro Cultures On Their Expressions

BackgroundThe developmental programme of embryo is controlled by genetic and epigenetic mechanisms. Epigenetics is used to describe the study of heritable changes in gene expression that occurs without a change in DNA sequence. During the two critical periods—gametogenesis and preimplantation development, there are genome-wide epigenetic reprogramming in gametes and embryos, which plays important roles in the erasion or creation of imprinting, gene silencing and tissue-specific gene expression.In eukaryotic cells, the fundamental unit of chromatin is nucleosome, which is composed of a histone octamer with two copies of H2A, H2B, H3 and H4 and 146 base pairs of DNA. The N-terminal tails of core histones are exposed on the nucleosome surface and can be modified by acetylation, phosphorylation, methylation, ubiquitination of specific amino acids. Of those post-translational modifications, acetylation occurs most frequently, that can regulate gene expressions by changing the high-order structures of chromatins. Hyper-acetylation or hypo-acetylation is related to transcription activation or repression. In vivo, thedynamic histone acetylation is controlled by histone acetyltransferases (HATs) and hsitone deacetylases (HDACs).Histone acetyltransferase GCN5 (GCN5) and histone deacetylase 1 (HDAC1) are the typical enzymes of HATs and HDACs. GCN5 is the transcriptional-related HATs and catalyze the acetylation of nucleosomal in nuclei or free histones in cytoplasm. Embryos die between 10 and 11 days post coitus in knock-out GCN5 gene mouse due to higher level of cell apoptosis. HDAC1 is one of RPD3-like histone deacetylases. It was first identified as an IL-2 inducible gene in mouse and can reflect the cell proliferation activity. GCN5 acts as transcriptional co-activator while HDAC1 as transcriptional co-repressor in gene expression. To summarize. GCN5 and HDAC1 are crucial to epigenetic reprogramming, regulation of gene expression and cell proliferation during embryo development. But to date, there is no report about GCN5 expression pattern in mouse preimplantational embryos, and only the study of HDAC1 expression in-vivo embryos has been reported.Assisted reproductive techniques (ARTs), such as in vitro fertilization and embryo transfer (IVF-ET), are very efficient in the treatment of infertility. It has been reported that in-vitro culture environments change the stage- and tissue-specific gene expression patterns of preimplantation embryos, such as the biallelic expression of imprinted genes and the levels of insulin-like growth factor 1(IGF-1), platelet-activating factor (PAF) and so on. However, few studies about the causes or mechanisms of them have been carried out.To investigate effects of in-vitro cultures on epigenetic reprogramming of preimplantational embryos and the possible mechanisms of abnormal expression patterns of some genes in in-vitro cultured embryos, we observed GCN5 and HDAC1 expression patterns in the mouse preimplantational embryos in vivo and their expression changes in in-vitro by means of reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry.ObjectiveThe aims of the present study are to study the expression pattern of GCN5 and HDAC1 in preimplantational embryos and the effects of in-vitro cultures on those enzymes expression, to investigate the influences of in-vitro cultures on epigenetic reprogramming of preimplantational embryos and the possible mechanisms of abnormal expression patterns of some genes in in-vitro cultured embryos.Material & methodsObjects of this study were ICR mouse preimplantational embryos.1. In-vivo group, ICR female mice were superovulated by Pregnant Mares' Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (HCG), and caged with stud males overnight. Embryos at the stage of two-cell, four-cell, eight-cell, morula, and blastocyst were retrieved directly from oviducts or uterus of pregnant mouse at post HCG (pHCG) 43~44 h, 58~60 h, 64~66 h, 78~80 h, 96~100h.2. In in-vitro group, ICR females were superovulated as above. Pronucleus embryo...