Genome activation in bovine embryos: Review of the literature and new insights from RNA sequencing experiments

https://doi.org/10.1016/j.anireprosci.2014.05.016Get rights and content

Highlights

  • We review studies on bovine embryonic genome activation (EGA).

  • We provide new insights into EGA by RNA sequencing (RNA-Seq).

  • A gene ontology analysis of genes activated at the 8-cell, 16-cell, and blastocyst stage is provided.

  • An outlook into potential applications of single cell RNA-Seq is given.

Abstract

Maternal-to-embryonic transition (MET) is the period in early embryonic development when maternal RNAs and proteins stored in the oocyte are gradually degraded and transcription of the embryonic genome is activated. First insights into the timing of embryonic genome activation (EGA) came from autoradiographic analyses of embryos following incorporation of [3H]uridine. These studies identified the eight- to 16-cell stage of bovine embryos as the period of major EGA, but detected first transcriptional activity already in one-cell embryos. Subsequent studies compared the transcriptome profiles of untreated embryos and of embryos incubated with the transcription inhibitor α-amanitin to reveal transcripts of embryonic origin. In addition, candidate gene-based and global gene expression studies over several stages of early development were performed and characteristic profiles were revealed. However, the onset of embryonic transcription was obscured by the presence of maternal transcripts and could only be determined for genes which are not expressed in oocytes. Using RNA sequencing of bovine germinal vesicle and metaphase II oocytes, and of four-cell, eight-cell, 16-cell and blastocyst stage embryos, we established the most comprehensive transcriptome data set of bovine oocyte maturation and early development. EGA was analyzed by (i) detection of embryonic transcripts which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. Using these three approaches we were able to map the onset of embryonic transcription for almost 7400 genes. Genes activated at the four-cell stage or before were functionally related to RNA processing, translation, and transport, preparing the embryo for major EGA at the eight-cell stage, when genes from a broad range of functional categories were found to be activated. These included transcriptional and translational functions as well as protein ubiquitination. The functions of the genes activated at the 16-cell stage were consistent with ongoing transcription and translation, while the genes activated in blastocysts included regulators of early lineage specification. Fine mapping of EGA provides a new layer of information for detecting disturbances of early development due to genetic, epigenetic, and environmental factors.

Introduction

The fusion of a male and a female gamete gives rise to an embryo. Initiation of development and the early embryonic developmental program is controlled by maternal transcripts and proteins produced and stored during oogenesis (reviewed in Tadros and Lipshitz, 2009). In the mouse a number of so-called maternal effect genes have been discovered which are transcribed during oogenesis. Functions of their products include processing of the male genome after fertilization, degradation of maternal RNAs and proteins, and the activation of the embryonic genome (reviewed in Li et al., 2010). As development proceeds, control is switched from maternal to embryo-derived transcripts and proteins. This crucial process in development has been termed maternal-to-embryonic transition (MET) and involves the following events: depletion of maternal transcripts by degradation and translation; replacement of maternal transcripts stored in oocytes by embryonic transcripts, e.g. ribosomal RNAs; and the generation of new embryo-specific transcripts (reviewed in Sirard, 2010). In zebrafish and rainbow trout, specific microRNAs (miRNAs) produced by the embryo have been shown to be involved in the destruction of maternal transcripts (Giraldez et al., 2006, Ramachandra et al., 2008). A role of specific miRNAs in MET was also suggested for bovine embryos. Mondou et al. (2012) observed an increase in the abundance of the mature forms of miR-130a and miR-21 and of the precursor form of miR-130a from the one-cell to the eight-cell stage, correlated with MET. Transcriptional inhibition of two-cell embryos by exposure to α-amanitin decreased the abundances of miR-21, pre-miR-21, and miR-130a, suggesting that these miRNAs were – at least in part – of embryonic origin. The authors suggested that miR-21 and miR-130a are involved in gene regulation during MET and may play a role in the degradation of maternal mRNAs. Another miRNA, which was found increased in abundance from the two-cell to the eight-cell stage of bovine embryogenesis, is miR-212. It was suggested as a negative regulator of maternal factor in the germ line alpha (FIGLA) transcripts during MET in bovine embryos (Tripurani et al., 2013). Other factors involved in the clearance of maternal transcripts during early development of metazoan embryos include RNA-binding proteins acting as specificity factors to direct the maternal degradation machinery to target mRNAs; signaling pathways that trigger production and/or activation of the clearance mechanism in early embryos; and mechanisms for spatial control of transcript clearance (reviewed in Walser and Lipshitz, 2011).

During MET, nuclear reprogramming is required to activate the transcriptionally inactive embryonic genome, which lasts two hours in Drosophila and takes one, three, and up to 3000 cell cycles in mouse, bovine and Xenopus embryos, respectively (reviewed in Sirard, 2010). Oocyte-stored products play an essential role in this process by altering the chromatin structure (Ostrup et al., 2013). The chromatin structure of early embryos, which impacts gene expression, can be altered by epigenetic modifications of DNA and histone proteins (Dean et al., 2001, Santos et al., 2003, Lepikhov et al., 2008, Wossidlo et al., 2011). Alterations in chromatin structure modulate the activity of transcription factors by permitting or restricting their access to regulatory elements of the genome, but are solely not sufficient to activate transcription. The oocyte cytoplasm plays also an important role in transcription activation by providing active transcription factors and RNA polymerase II (reviewed in Kanka, 2003).

The initiation of gene expression largely based on the products of an embryo is referred to as embryonic genome activation (EGA) and, as a part of MET, is the most important event in the pre-implantation development of mammals. The mechanisms regulating the onset of EGA are thought to be broadly conserved in mammals, despite species-specific differences in the timing of major EGA which ranges from the two-cell stage in mouse embryos (reviewed in Wang and Dey, 2006) to the four- to eight-cell stage in human (Braude et al., 1988) and pig embryos (reviewed in Sirard, 2012), and the eight- to 16-cell stage in bovine and rabbit embryos (Telford et al., 1990, Sirard, 2012). Cell cycle chronology, which is species-specific, and a cell cycle-dependent localization of RNA polymerase II in the nuclei are probably related to embryonic transcription (Marcucio et al., 1995) and may, at least partly, account for the differences in the onset of EGA.

EGA appears to start gradually and is preceded by an initial minor embryonic transcription. Gene expression studies during preimplantation mouse embryo development revealed three successive, overlapping waves of gene expression corresponding to minor EGA (one-cell stage), major EGA (two- to four-cell stage), and mid-preimplantation gene activation (MGA; four- to eight-cell stage). Subsequent waves of gene expression were found to be associated with morula compaction and blastocyst cavitation (reviewed in Wang and Dey, 2006). Genes involved in cell proliferation, mitotic cell cycle, regulation of transcription, DNA and protein metabolism were found to be early expressed (Kanka et al., 2012). Although specific mechanisms of the initiation of EGA remain to be elucidated, the involvement of some factors like maternal cyclin A2 (CCNA2), retinoblastoma protein (RB1), catalytic subunit of SWI/SNF related chromatin remodeling complex (BRG1) and sex determining region Y-box2 (SOX2), was recently suggested in a model of EGA in mouse (Kanka et al., 2012).

In bovine embryos, major EGA has been described to occur at the eight- to 16-cell stage, but the onset of EGA has not been precisely defined and varied dependent on the respective techniques used for detecting embryonic transcription. Using [3H]uridine incorporation after short incubation as an indicator, EGA in bovine embryos appeared to occur at the eight- to 16-cell stage (Camous et al., 1986, Frei et al., 1989). A further evidence for bovine EGA at the eight-cell stage was given by a study using polypeptide profiles of bovine in vitro embryos treated with α-amanitin (Barnes and First, 1991). The authors showed that these embryos were able to develop only up to the eight-cell stage, indicating the requirement of embryonic transcripts for further development at this stage. The eight-cell stage was also characterized by major changes in the structure of blastomere nucleoli, i.e. nucleolus precursor bodies (NPBs), electron-dense spherical masses of tightly packed fibrils, transformed into a fibrillo-granular nucleolus including formation of primary eccentric and secondary peripheral vacuoles (reviewed in Svarcova et al., 2007). However, after long-term exposure to [3H]uridine, transcriptional activity could be already detected in two- to four-cell (Plante et al., 1994, Hyttel et al., 1996, Viuff et al., 1996, Memili et al., 1998) or even in one-cell bovine embryos (Memili and First, 1999), suggesting minor EGA already at these early stages of development. First ribosomal RNA transcription was visualized in four-cell embryos using a combination of fluorescence in situ hybridization (FISH) and silver staining (Viuff et al., 1998).

Subsequent studies aimed at the characterization of EGA in bovine embryos by using experimental approaches combining incubation with the transcription inhibitor α-amanitin and subsequent expression profiling by reverse transcriptase-polymerase chain reaction (RT-PCR) or array hybridization-based methods and are reviewed below.

Section snippets

Insights into EGA by RT-PCR and microarray studies

To investigate the onset of EGA with a resolution higher than global transcription initiation, bovine embryos from the one-cell to the expanded/hatched blastocyst stage were cultured with and without α-amanitin for 4 or 12 h, and stage-specific α-amanitin sensitive transcripts were evaluated by differential display (DD)-RT-PCR (Natale et al., 2000). Sensitivity of the DD-RT-PCR band pattern to α-amanitin was first detected at the two- to five-cell stage but became predominant following the six-

RNA sequencing for the study of early bovine development

RNA sequencing (RNA-Seq) has a number of advantages as compared to hybridization-based techniques, such as microarrays, which compare only relative transcript abundances (reviewed in Wang et al., 2009). RNA-Seq enables the direct determination of the cDNA sequences from millions of short fragments, allowing transcriptome analyses at single nucleotide resolution. As compared to hybridization-based techniques, RNA-Seq has a higher sensitivity, a higher dynamic range, and less background (Wang et

Strategies to identify EGA by RNA sequencing

In our RNA-Seq study of bovine oocytes (GV and MII) and early embryos (four-cell, eight-cell, 16-cell, and blastocyst) we used three different strategies to fine-map EGA (Graf et al., 2014): (i) detection of embryonic transcripts not present in oocytes; (ii) detection of paternal specific SNPs as a marker for the onset of EGA; and (iii) detection of incompletely spliced transcripts as an indicator of de novo transcription.

The most elementary approach to look for newly expressed genes during EGA

Relevance and outlook

In our study (Graf et al., 2014), we performed RNA-Seq analyses of pools of in vitro produced bovine embryos. These are known to be developmentally less competent than their in vivo derived counterparts (reviewed in Lonergan and Fair, 2008). Therefore it would be most interesting to repeat the RNA-Seq experiments with in vivo derived embryos, which might provide new molecular insights into the developmental differences of in vitro vs. in vivo derived embryos. Another question is how different

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

Our studies of bovine embryonic genome activation were supported by the EU grants Plurisys (HEALTH-F4-2009-223485 FP7 Health 534 project) and Fecund (FP7-KBBE-2012-6 project 312097), by the Deutsche Forschungsgemeinschaft (FOR 1041), and by BioSysNet. MHB is supported by a DFG fellowship through QBM.

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