Time Of Embryonic Genome Activation Research Articles

Genome-wide methylation profile of mitochondrial DNA across bovine preimplantation development

ABSTRACT This study characterized variations in the methylation profile of mitochondrial DNA (mtDNA) during initial bovine embryo development and correlated the presence of methylation with mtDNA transcription. Bovine oocytes were obtained from abattoir ovaries and submitted to in vitro culture procedures. Oocytes and embryos were collected at various stages (immature oocyte, IM; mature oocyte, MII; zygote, ZY; 4-cells, 4C; 16-cells, 16C and blastocysts, BL). Total DNA (including mtDNA) was used for Whole Genome Enzymatic Methyl Sequencing and for quantification of mtDNA copy number. Extracted RNA was used for quantification of mitochondrial transcripts using Droplet Digital PCR. We selected ND6, CYTB, tRNA-Phe and tRNA-Gln based on their location in the mitochondrial genome, functionality and/or previous literature associating these regions with cytosine methylation. The number of mtDNA copies per oocyte/embryo was found to be similar, while methylation levels in mtDNA varied among stages. Higher total methylation levels were found mainly at 4C and 16C. In specific gene regions, higher methylation levels were also observed at 4C and 16C (ND6, CYTB and tRNA-Phe), as well as an inverse correlation with the quantity of transcripts for these regions. This is a first description of epigenetic changes occurring in mtDNA during early embryonic development. Our results indicate that methylation might regulate the mtDNA transcription at a local level, particularly around the time of embryonic genome activation.

The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo

BackgroundDespite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro-derived bovine embryos, there is no comparison at the protein expression level.ResultsA total of 38 pools of grade-1 quality bovine embryos at the 4–6 cell, 8–12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development.ConclusionsThe maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4–6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

Gene expression at different cell stages of in vitro-fertilized bovine embryos

The birth rate of embryos produced in vitro (IVF) is still lower than that of embryos produced in vivo. Three major steps for the success of the IVF technique are maturation of immature oocytes, fertilization of matured oocytes, and culture of the resulting embryos. Studying mRNA expression in early embryonic development stages is important and can help to assess embryo quality and optimize production protocols in vitro. The current study aimed to determine the expression levels of developmentally important genes in different stages of bovine embryos produced in vitro. Cumulus-oocyte complexes (COCs) were collected from bovine ovaries and cultured in synthetic oviduct fluid (SOF) medium for 7 - 9 days. Embryos were collected at the time-points listed above, and mRNA expression of genes involved in pluripotency (OCT4), DNA methylation (DNMT1), apoptosis (BAX), and metabolism (GLUT1) and a heat shock protein (HSP70) was estimated from the 2-cell stage to the blastocyst stage of embryos. The results showed statistically significant differences in the relative abundance (RA) of OCT4, DNMT1, BAX, and GLUT1 gene transcripts among the different stages, whereas there were non-significant differences in the RA of HSP70 between these stages. In conclusion, gene expression levels differ among the developmental stages of embryos produced in vitro, possibly because of the timing of embryonic genome activation (EGA).

Dynamic Changes in the Proteome of Early Bovine Embryos Developed In Vivo.

Early embryo development is a dynamic process involving important molecular and structural changes leading to the embryonic genome activation (EGA) and early cell lineage differentiation. Our aim was to elucidate proteomic changes in bovine embryos developed in vivo. Eleven females were used as embryo donors and pools of embryos at the 4–6 cell, 8–12 cell, morula, compact morula and blastocyst stages were analyzed by nanoliquid chromatography coupled with label free quantitative mass spectrometry. A total of 2,757 proteins were identified, of which 1,950 were quantitatively analyzed. Principal component analysis of data showed a clear separation of embryo pools according to their developmental stage. The hierarchical clustering of differentially abundant proteins evidenced a first cluster of 626 proteins that increased in abundance during development and a second cluster of 400 proteins that decreased in abundance during development, with most significant changes at the time of EGA and blastocyst formation. The main pathways and processes overrepresented among upregulated proteins were RNA metabolism, protein translation and ribosome biogenesis, whereas Golgi vesicle transport and protein processing in endoplasmic reticulum were overrepresented among downregulated proteins. The pairwise comparison between stages allowed us to identify specific protein interaction networks and metabolic pathways at the time of EGA, morula compaction and blastocyst formation. This is the first comprehensive study of proteome dynamics in non-rodent mammalian embryos developed in vivo. These data provide a number of protein candidates that will be useful for further mechanistic and functional studies.

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

BackgroundThe transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.ResultsIn vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14–3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.ConclusionsOur findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Chromatin remodeling in bovine embryos indicates species-specific regulation of genome activation

The shift from maternal to embryonic control is a critical developmental milestone in preimplantation development. Widespread transcriptomic and epigenetic remodeling facilitate this transition from terminally differentiated gametes to totipotent blastomeres, but the identity of transcription factors (TF) and genomic elements regulating embryonic genome activation (EGA) are poorly defined. The timing of EGA is species-specific, e.g., the timing of murine and human EGA differ significantly. To deepen our understanding of mammalian EGA, here we profile changes in open chromatin during bovine preimplantation development. Before EGA, open chromatin is enriched for maternal TF binding, similar to that observed in humans and mice. During EGA, homeobox factor binding becomes more prevalent and requires embryonic transcription. A cross-species comparison of open chromatin during preimplantation development reveals strong similarity in the regulatory circuitry underlying bovine and human EGA compared to mouse. Moreover, TFs associated with murine EGA are not enriched in cattle or humans, indicating that cattle may be a more informative model for human preimplantation development than mice.

59 Genome activation in intracytoplasmic sperm injection-derived horse embryos

During pre-implantation development, embryos go through a critical period of embryonic genome activation (EGA). The timing of EGA is species specific, but little is known in horse embryos. Here, we aimed to characterise EGA in equine embryos produced by intracytoplasmic sperm injection. Embryos were produced by intracytoplasmic sperm injection of oocytes from 3 mares. Two embryos from each mare at each of 8 developmental stages (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst) were individually analysed by RNA-seq. Differential expression was evaluated using binomial Wald tests with an absolute logFC (fold change) threshold of 1 in the DESEqn 2R package. We found that EGA occurred in a two-step fashion. Minor EGA took place during the 2-cell to 4-cell transition, and featured up-regulation of 751 genes and discrete down-regulation of 60 genes in 4-cell embryos compared with 2-cell embryos. Differentially upregulated genes were enriched in gene ontology terms related to transcriptional activator activity, homeobox domains, and nucleosome assembly. Major EGA occurred during the 4-cell to 8-cell transition and included the largest number of differentially expressed genes (n=2,023) between consecutive stages. This period also featured the first massive transcript downregulation (n=816). Upregulated genes were enriched in gene ontology terms related to ribosomal assembly, translation, and RNA modification. Additionally, we observed that the number of intronic sequences was significantly higher from the 4-cell stage onward, indicating active transcription in comparison to oocytes, zygotes, and 2-cell embryos. To evaluate the timing of paternal genome activation, we used whole-genome sequencing data from the parents (average genome coverage of 19×) to quantify allele-specific expression. The average number of informative SNPs in exons, i.e. SNPs with alternative homozygous genotypes from the sire (AA mare - BB sire), was 26 128 per mare, corresponding to 7696 genes. Parental-specific transcript abundance was determined for each embryo, with an average of 1,911±865 informative SNPs detected per sample. Paternal alleles were considered expressed when they reached 10% of the maternal count. Across development, paternal transcripts became appreciable at the 4-cell stage, with 14.15±7.60% of the informative SNPs exhibiting paternal expression, and increased thereafter until reaching a maximum of 96.34% at the blastocyst stage. Overall, this work demonstrates that EGA in horse embryos starts at the 4-cell stage and achieves its main activation at the 8-cell stage. Further analysis will be performed to detail paternal vs. maternal gene expression at the different embryonic stages.

DNA methylation and miRNA-1296 act in concert to mediate spatiotemporal expression of KPNA7 during bovine oocyte and early embryonic development

BackgroundEpigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and microRNA (miRNA)-mediated mRNA degradation of KPNA7 were examined.ResultsComparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2′-deoxycytidine (5-Aza-CdR). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza-CdR showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR (RT-qPCR) analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression.ConclusionsThese results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition.

Systems based analysis of human embryos and gene networks involved in cell lineage allocation

BackgroundLittle is understood of the molecular mechanisms involved in the earliest cell fate decision in human development, leading to the establishment of the trophectoderm (TE) and inner cell mass (ICM) stem cell population. Notably, there is a lack of understanding of how transcriptional networks arise during reorganisation of the embryonic genome post-fertilisation.ResultsWe identified a hierarchical structure of preimplantation gene network modules around the time of embryonic genome activation (EGA). Using network models along with eukaryotic initiation factor (EIF) and epigenetic-associated gene expression we defined two sets of blastomeres that exhibited diverging tendencies towards ICM or TE. Analysis of the developmental networks demonstrated stage specific EIF expression and revealed that histone modifications may be an important epigenetic regulatory mechanism in preimplantation human embryos. Comparison to published RNAseq data confirmed that during EGA the individual 8-cell blastomeres are transcriptionally primed for the first lineage decision in development towards ICM or TE.ConclusionsUsing multiple systems biology approaches to compare developmental stages in the early human embryo with single cell transcript data from blastomeres, we have shown that blastomeres considered to be totipotent are not transcriptionally equivalent. Furthermore we have linked the developmental interactome to individual blastomeres and to later cell lineage. This has clinical implications for understanding the impact of fertility treatments and developmental programming of long term health.

Oxidative stress in sperm affects the epigenetic reprogramming in early embryonic development

BackgroundReactive oxygen species (ROS)-induced oxidative stress is well known to play a major role in male infertility. Sperm are sensitive to ROS damaging effects because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. However, how oxidative DNA lesions in sperm affect early embryonic development remains elusive.ResultsUsing cattle as model, we show that fertilization using sperm exposed to oxidative stress caused a major developmental arrest at the time of embryonic genome activation. The levels of DNA damage response did not directly correlate with the degree of developmental defects. The early cellular response for DNA damage, γH2AX, is already present at high levels in zygotes that progress normally in development and did not significantly increase at the paternal genome containing oxidative DNA lesions. Moreover, XRCC1, a factor implicated in the last step of base excision repair (BER) pathway, was recruited to the damaged paternal genome, indicating that the maternal BER machinery can repair these DNA lesions induced in sperm. Remarkably, the paternal genome with oxidative DNA lesions showed an impairment of zygotic active DNA demethylation, a process that previous studies linked to BER. Quantitative immunofluorescence analysis and ultrasensitive LC–MS-based measurements revealed that oxidative DNA lesions in sperm impair active DNA demethylation at paternal pronuclei, without affecting 5-hydroxymethylcytosine (5hmC), a 5-methylcytosine modification that has been implicated in paternal active DNA demethylation in mouse zygotes. Thus, other 5hmC-independent processes are implicated in active DNA demethylation in bovine embryos. The recruitment of XRCC1 to damaged paternal pronuclei indicates that oxidative DNA lesions drive BER to repair DNA at the expense of DNA demethylation. Finally, this study highlighted striking differences in DNA methylation dynamics between bovine and mouse zygotes that will facilitate the understanding of the dynamics of DNA methylation in early development.ConclusionsThe data demonstrate that oxidative stress in sperm has an impact not only on DNA integrity but also on the dynamics of epigenetic reprogramming, which may harm the paternal genetic and epigenetic contribution to the developing embryo and affect embryo development and embryo quality.

Three-dimensional analysis of nuclear heterochromatin distribution during early development in the rabbit

Changes to the spatial organization of specific chromatin domains such as constitutive heterochromatin have been studied extensively in somatic cells. During early embryonic development, drastic epigenetic reprogramming of both the maternal and paternal genomes, followed by chromatin remodeling at the time of embryonic genome activation (EGA), have been observed in the mouse. Very few studies have been performed in other mammalian species (human, bovine, or rabbit) and the data are far from complete. During this work, we studied the three-dimensional organization of pericentromeric regions during the preimplantation period in the rabbit using specific techniques (3D-FISH) and tools (semi-automated image analysis). We observed that the pericentromeric regions (identified with specific probes for Rsat I and Rsat II genomic sequences) changed their shapes (from pearl necklaces to clusters), their nuclear localizations (from central to peripheral), as from the 4-cell stage. This reorganization goes along with histone modification changes and reduced amount of interactions with nucleolar precursor body surface. Altogether, our results suggest that the 4-cell stage may be a crucial window for events necessary before major EGA, which occurs during the 8-cell stage in the rabbit.

H3K9 demethylase KDM4E is an epigenetic regulator for bovine embryonic development and a defective factor for nuclear reprogramming.

Aberrant epigenetic reprogramming often results in developmental defects in somatic cell nuclear transfer (SCNT) embryos during embryonic genome activation (EGA). Bovine eight-cell SCNT embryos exhibit global hypermethylation of histone H3 lysine 9 tri- and di-methylation (H3K9me3/2), but the intrinsic reason for this remains elusive. Here, we provide evidence that two H3K9 demethylase genes, lysine-specific demethylase 4D (KDM4D) and 4E (KDM4E), are related to active H3K9me3/2 demethylation in in vitro fertilized (IVF) embryos and are deficiently expressed in cloned embryos at the time of EGA. Moreover, KDM4E plays a more crucial role in IVF and SCNT embryonic development, and overexpression of KDM4E can restore the global transcriptome, improve blastocyst formation and increase the cloning efficiency of SCNT embryos. Our results thereby indicate that KDM4E can function as a crucial epigenetic regulator of EGA and as an internal defective factor responsible for persistent H3K9me3/2 barriers to SCNT-mediated reprogramming. Furthermore, we show that interactions between RNA and KDM4E are essential for H3K9 demethylation during EGA. These observations advance the understanding of incomplete nuclear reprogramming and are of great importance for transgenic cattle procreation.

35 Interspecies Somatic Cell Nuclear Transfer Embryos that Form Nucleoli do not Always Activate Mitochondrial Functional Differentiation at the Time of Embryonic Genome Activation

Embryonic genome activation (EGA) is a complex process that needs a good orchestration of all biochemical processes at the time of maternal-to-embryonic transition. Mitochondria are strictly dependent on the nucleus for their correct activity as ~1500 mitochondrial genes have nuclear localisation. The finding of transcriptional activation and accumulation of mRNAs related to mitochondrial biogenesis (Mtango et al. 2008 Reprod. Fertil. Dev. 20, 846-859) around the time of EGA confirmed the role of nucleus in this process. Studying mitochondria behaviour in interspecies somatic cell nuclear transfer (iSCNT) embryos (Lagutina et al. 2010 Reproduction 140, 273-285), we have found that at the time of EGA, mitochondria activation could be demonstrated by JC-1 accumulation. We suggested that comparison of the mean green fluorescence intensity (FI) that corresponds to the fluorescence of the monomeric form of the dye and correlates to relative mitochondrial mass (Mancini et al. 1997) in iSCNT and control nuclear transfer (NT) could serve as a test to assess EGA in iSCNT embryos. The aim of this study was to estimate nuclear-cytoplasmic interaction in iSCNT embryos that formed nucleoli at the time of EGA (Lagutina et al. 2011 Reproduction 141, 453-465) such as embryos derived from bovine oocytes and bovine (control), buffalo or ovine donor nuclei, and from porcine oocytes and porcine (control), horse, or rabbit nuclei. Embryos 72 h after activation were stained with 2 μM JC-1 in SOF-HEPES with 10% FCS at 37°C for 1 h. Images were collected using a fluorescein isothiocyanate (FITC) filter and analysed with Adobe Photoshop Elements 2 (Adobe Systems, San Jose, CA, USA). The data are presented as mean FI of the embryo. To demonstrate the effect of EGA inhibition on mitochondria, bovine and porcine NT embryos were cultured in medium supplemented with 25 μg/mL α-amanitin (AA) from 48 to 72 h after activation. The analyses of mean FI of the embryos showed that ovine and buffalo nuclei were able to support mitochondrial mass accumulation in iSCNT embryos with bovine oocytes equal to control bovine NT embryos (35 ± 11.2; 41.9 ± 14.8; 36.2 ± 7.6, respectively) that was significantly higher than in bovine embryos treated with AA (15.4 ± 4.9; P < 0.05). In the iSCNT embryos composed of porcine oocytes and equine or rabbit nuclei, mean FI values (20 ± 13.4; 18.3 ± 5.5, respectively) were comparable with those in porcine NT embryos treated with AA (16.2 ± 6.2), and were significantly lower than in porcine control (91 ± 47.7; P < 0.05) NT embryos, demonstrating the inability of equine and rabbit nuclei to properly govern the porcine mitochondria mass growth at the time of EGA. In conclusion, nucleolus formation and activation of nuclear encoded mitochondrial genes at the time of EGA cannot serve, per se, as a marker of correct embryonic genome activation in iSCNT embryos, because, in our conditions, no iSCNT embryos developed to blastocyst (Lagutina et al. 2010 Reproduction 140, 273-285). This knowledge about behaviour of different embryo compartments at the time of EGA could extend our understanding of the whole process. This work was funded by Translink (EU FP7 no. 603049) and Xenoislet projects (EU FP7 no. 601827).

Altered expression of BRG1 and histone demethylases, and aberrant H3K4 methylation in less developmentally competent embryos at the time of embryonic genome activation.

Epigenetics is a fundamental regulator underlying many biological functions, such as development and cell differentiation. Epigenetic modifications affect key chromatin regulation, including transcription and DNA repair, which are critical for normal embryo development. In this study, we profiled the expression of epigenetic modifiers and patterns of epigenetic changes in porcine embryos around the period of embryonic genome activation (EGA). We observed that Brahma-related gene 1 (BRG1) and Lysine demethylase 1A (KDM1A), which can alter the methylation status of lysine 4 in histone 3 (H3K4), localize to the nucleus at Day 3-4 of development. We then compared the abundance of epigenetic modifiers between early- and late-cleaving embryos, which were classified based on the time to the first cell cleavage, to investigate if their nuclear localization contributes to developmental competence. The mRNA abundance of BRG1, KDM1A, as well as other lysine demethylases (KDM1B, KDM5A, KDM5B, and KDM5C), were significantly higher in late- compared to early-cleaving embryos near the EGA period, although these difference disappeared at the blastocyst stage. The abundance of H3K4 mono- (H3K4me) and di-methylation (H3K4me2) during the EGA period was reduced in late-cleaving and less developmentally competent embryos. By contrast, BRG1, KDM1A, and H3K4me2 abundance was greater in embryos with more than eight cells at Day 3-4 of development compared to those with fewer than four cells. These findings suggest that altered epigenetic modifications of H3K4 around the EGA period may affect the developmental capacity of porcine embryos to reach the blastocyst stage. Mol. Reprod. Dev. 84: 19-29, 2017. © 2016 Wiley Periodicals, Inc.

The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

Abstract One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos

Dppa3 has been described in mice as an important maternal factor contributed by the oocyte that participates in protecting the maternal genome from oxidation of methylated cytosines (5mC) to hydroxymethylated cytosines (5hmC). Dppa3 is also required for normal mouse preimplantation development. This gene is poorly conserved across mammalian species, with less than 32% of protein sequence shared between mouse, cow and human. RNA-seq analysis of bovine oocytes and preimplantation embryos revealed that DPPA3 transcripts are some of the most highly abundant mRNAs in the oocyte, and their levels gradually decrease toward the time of embryonic genome activation (EGA). Knockdown of DPPA3 by injection of siRNA in germinal vesicle (GV) stage oocytes was used to assess its role in epigenetic remodeling and embryo development. DPPA3 knockdown resulted in increased intensity of 5hmC staining in the maternal pronucleus (PN), demonstrating a role for this factor in the asymmetric remodeling of the maternal and paternal PN in bovine zygotes. Also, DPPA3 knockdown decreased the developmental competence of parthenogenetic and in vitro fertilized embryos. Finally, DPPA3 knockdown embryos that reached the blastocyst stage had significantly fewer ICM cells as compared with control embryos. We conclude that DPPA3 is a maternal factor important for correct epigenetic remodeling and normal embryonic development in cattle, indicating that the role of DPPA3 during early development is conserved between species.

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

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.

Metabolic heterogeneity during preimplantation development: the missing link?

Most tissues in the body rely on the presence of gap junctions in order to couple their component cells electrically and metabolically via intercellular transport of ions, metabolites and signalling agents. As a result, cells within tissues achieve a high degree of, 'metabolic homogeneity' which enables them to develop in an integrated way and co-ordinate their response to physiological signals and environmental cues. Unusually, the developing mammalian preimplantation embryo does not form functional gap junctions until it has divided into 8 or more cells. We discuss the implications of this 'missing link' during the first few days of development for the maintenance of homogeneity between embryonic cells and for the co-ordination of the embryonic response to intrinsic genetic damage and external environmental signals. No systematic review has been carried out. The physiology of preimplantation development and the general nature of gap junctions have been reviewed briefly before examining experimental evidence which addresses the following points: (i) whether there are functional differences between early blastomeres; (ii) when during preimplantation development the embryo is most sensitive to environmental perturbation and (iii) the consequences for early embryos of ablating gap junction formation and function. General conclusions are confounded by species differences, especially in the timing of embryonic genome activation (EGA) and the extent of intrinsic genotypic and phenotypic variation (low in embryos from inbred mice; high in human embryos). Nevertheless, we propose that the absence of gap junctions requires cleavage stage mammalian embryos to behave cell autonomously in a metabolic sense, contributes to their heightened sensitivity to environmental perturbation compared with the later stages of preimplantation development and poses more problems in the early human embryo, where there is a high degree of heterogeneity between the blastomeres. We argue that the legacy of metabolic heterogeneity, in part generated by the absence of gap junctions, is 'rescued' by the onset of apoptosis following EGA. In the context of human-assisted conception, since early embryos lacking gap junctions are more sensitive to environmental stress during cleavage, this would support transfer to the natural environment as early as possible after fertilization.

Transcriptomic signature to oxidative stress exposure at the time of embryonic genome activation in bovine blastocysts

In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions. Two pro-oxidant agents, one that acts extracellularly by promoting reactive oxygen species (ROS) production (0.01 mM 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) or another that acts intracellularly by inhibiting glutathione synthesis (0.4 mM buthionine sulfoximine [BSO]) were added separately to in vitro culture media from Day 3 (8-16-cell stage) onward. Transcriptomic analysis was then performed on resulting Day-7 blastocysts. In the literature, these two pro-oxidant conditions were shown to induce delayed degeneration in a proportion of Day-8 blastocysts. In our experiment, no morphological difference was visible, but AAPH tended to decrease the blastocyst rate while BSO significantly reduced it, indicating a differential impact on the surviving population. At the transcriptomic level, blastocysts that survived either pro-oxidant exposure showed oxidative stress and an inflammatory response (ARRB2), although AAPH induced higher disturbances in cellular homeostasis (SERPINE1). Functional genomics of the BSO profile, however, identified differential expression of genes related to glycine metabolism and energy metabolism (TPI1). These differential features might be indicative of pre-degenerative blastocysts (IGFBP7) in the AAPH population whereas BSO exposure would select the most viable individuals (TKDP1). Together, these results illustrate how oxidative disruption of pre-attachment development is associated with systematic up-regulation of several metabolic markers. Moreover, it indicates that a better capacity to survive anti-oxidant depletion may allow for the survival of blastocysts with a quieter metabolism after compaction.

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.