Several lines of evidence indicate that the formation of a transcriptionally repressive state during the two-cell stage in the preimplantation mouse embryo is superimposed on the activation of the embryonic genome. However, it is difficult to determine the profile of newly synthesized (nascent) RNA during this phase because large amounts of maternal RNA accumulate in maturing oocytes to support early development. Using 5-bromouridine-5'-triphosphate labeling of RNA, we have verified that nascent RNA synthesis was repressed between the two-cell and four-cell transition in normally fertilized but not in parthenogenetic embryos. Moreover, this repression was contributed by sperm (male) chromatin, which we confirmed by studying androgenetic embryos. The source of factors responsible for repressing nascent RNA production was investigated using different stages of sperm development. Fertilization with immature round spermatids resulted in a lower level of transcriptional activity than with ICSI at the two-cell stage, and this was consistent with further repression at the four-cell stage in the ICSI group. Finally, study on DNA replication and chromatin remodeling was performed using labeled histones H3 and H4 to differentiate between male and female pronuclei. The combination of male and female chromatin appeared to decrease nascent RNA production in the fertilized embryo. This study indicates that paternal chromatin is important in the regulation of transcriptional activity during mouse preimplantation development and that this capacity is acquired during spermiogenesis.
"Reference genes were chosen from those used routinely in studies of pre-implantation embryonic stages
[11, 36, 37]. Other potentially suitable reference genes were selected among those used in published literature on the reproductive system (Table
[38–41]. Whenever possible, primers fulfilled the following recommended criteria: amplicon length of 80 bp – 130 bp, location of primers on two different exons, primer sequence length of 18–25 bp, melting temperature of 58°C ± 2°C and GC content of 40% – 60%. "
[Show abstract][Hide abstract] ABSTRACT: Background
Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied.
mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively.
These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-675) contains supplementary material, which is available to authorized users.
BMC Research Notes 09/2014; 7(1):675. DOI:10.1186/1756-0500-7-675
"In addition to polyadenylation controls (Oh et al. 2000; Schultz 2002), asRNAs may uncouple transcription from translation. Our analysis of differential gene expression demonstrated a transcriptionally repressed state during development (Fig. 3A), in agreement with previous reports (Schultz 2002; Bui et al. 2011). Therefore, gene silencing appears to be an essential mechanism in development , in a fashion similar to gene activation (Wang and Dey 2006). "
[Show abstract][Hide abstract] ABSTRACT: Fertilization precisely choreographs parental genomes by using gamete-derived cellular factors and activating genome regulatory programs. However, the mechanism remains elusive owing to the technical difficulties of preparing large numbers of high-quality preimplantation cells. Here, we collected >14 × 10(4) high-quality mouse metaphase II oocytes and used these to establish detailed transcriptional profiles for four early embryo stages and parthenogenetic development. By combining these profiles with other public resources, we found evidence that gene silencing appeared to be mediated in part by noncoding RNAs and that this was a prerequisite for post-fertilization development. Notably, we identified 817 genes that were differentially expressed in embryos after fertilization compared with parthenotes. The regulation of these genes was distinctly different from those expressed in parthenotes, suggesting functional specialization of particular transcription factors prior to first cell cleavage. We identified five transcription factors that were potentially necessary for developmental progression: Foxd1, Nkx2-5, Sox18, Myod1, and Runx1. Our very large-scale whole-transcriptome profile of early mouse embryos yielded a novel and valuable resource for studies in developmental biology and stem cell research. The database is available at http://dbtmee.hgc.jp.
Genes & Development 12/2013; 27(24):2736-48. DOI:10.1101/gad.227926.113 · 10.80 Impact Factor
"The conversion reaction in the paternal pronucleus starts at 4–6 h after fertilization, and a marked asymmetry in the male and female pronucleus is clearly evident 8 h after fertilization. The first DNA replication also starts in almost the same period in both pronuclei [29,30]. In an unpublished experimental result, the DNA synthesis started at 5–6 h after fertilization and terminated before 9 h (figure 2; T. Kohda 2012, unpublished data). "
[Show abstract][Hide abstract] ABSTRACT: The early stage of mammalian development from fertilization to implantation is a period when global and differential changes in the epigenetic landscape occur in paternally and maternally derived genomes, respectively. The sperm and egg DNA methylation profiles are very different from each other, and just after fertilization, only the paternally derived genome is subjected to genome-wide hydroxylation of 5-methylcytosine, resulting in an epigenetic asymmetry in parentally derived genomes. Although most of these differences are not present by the blastocyst stage, presumably due to passive demethylation, the maintenance of genomic imprinting memory and X chromosome inactivation in this stage are of critical importance for post-implantation development. Zygotic gene activation from paternally or maternally derived genomes also starts around the two-cell stage, presumably in a different manner in each of them. It is during this period that embryo manipulation, including assisted reproductive technology, is normally performed; so it is critically important to determine whether embryo manipulation procedures increase developmental risks by disturbing subsequent gene expression during the embryonic and/or neonatal development stages. In this review, we discuss the effects of various embryo manipulation procedures applied at the fertilization stage in relation to the epigenetic asymmetry in pre-implantation development. In particular, we focus on the effects of intracytoplasmic sperm injection that can result in long-lasting transcriptome disturbances, at least in mice.
Philosophical Transactions of The Royal Society B Biological Sciences 01/2013; 368(1609):20120353. DOI:10.1098/rstb.2012.0353 · 7.06 Impact Factor
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