Journal articles on the topic 'Zygote Genome Activation'
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1
Torres-Padilla, Maria Elena, and Magdalena Zernicka-Goetz. "Role of TIF1α as a modulator of embryonic transcription in the mouse zygote." Journal of Cell Biology 174, no. 3 (July 31, 2006): 329–38. http://dx.doi.org/10.1083/jcb.200603146.
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The first events of the development of any embryo are under maternal control until the zygotic genome becomes activated. In the mouse embryo, the major wave of transcription activation occurs at the 2-cell stage, but transcription starts already at the zygote (1-cell) stage. Very little is known about the molecules involved in this process. We show that the transcription intermediary factor 1 α (TIF1α) is involved in modulating gene expression during the first wave of transcription activation. At the onset of genome activation, TIF1α translocates from the cytoplasm into the pronuclei to sites of active transcription. These sites are enriched with the chromatin remodelers BRG-1 and SNF2H. When we ablate TIF1α through either RNA interference (RNAi) or microinjection of specific antibodies into zygotes, most of the embryos arrest their development at the 2–4-cell stage transition. The ablation of TIF1α leads to mislocalization of RNA polymerase II and the chromatin remodelers SNF2H and BRG-1. Using a chromatin immunoprecipitation cloning approach, we identify genes that are regulated by TIF1α in the zygote and find that transcription of these genes is misregulated upon TIF1α ablation. We further show that the expression of some of these genes is dependent on SNF2H and that RNAi for SNF2H compromises development, suggesting that TIF1α mediates activation of gene expression in the zygote via SNF2H. These studies indicate that TIF1α is a factor that modulates the expression of a set of genes during the first wave of genome activation in the mouse embryo.
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Ntostis, Panagiotis, Deborah Carter, David Iles, John Huntriss, Maria Tzetis, and David Miller. "Potential sperm contributions to the murine zygote predicted by in silico analysis." Reproduction 154, no. 6 (December 2017): 777–88. http://dx.doi.org/10.1530/rep-17-0097.
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Paternal contributions to the zygote are thought to extend beyond delivery of the genome and paternal RNAs have been linked to epigenetic transgenerational inheritance in different species. In addition, sperm–egg fusion activates several downstream processes that contribute to zygote formation, including PLC zeta-mediated egg activation and maternal RNA clearance. Since a third of the preimplantation developmental period in the mouse occurs prior to the first cleavage stage, there is ample time for paternal RNAs or their encoded proteins potentially to interact and participate in early zygotic activities. To investigate this possibility, a bespoke next-generation RNA sequencing pipeline was employed for the first time to characterise and compare transcripts obtained from isolated murine sperm, MII eggs and pre-cleavage stage zygotes. Gene network analysis was then employed to identify potential interactions between paternally and maternally derived factors during the murine egg-to-zygote transition involving RNA clearance, protein clearance and post-transcriptional regulation of gene expression. Ourin silicoapproach looked for factors in sperm, eggs and zygotes that could potentially interact co-operatively and synergistically during zygote formation. At least five sperm RNAs (Hdac11,Fbxo2,Map1lc3a,Pcbp4andZfp821) met these requirements for a paternal contribution, which with complementary maternal co-factors suggest a wider potential for extra-genomic paternal involvement in the developing zygote.
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Lee, Hyeonji, Seong-Yeob You, Dong Wook Han, Hyeonwoo La, Chanhyeok Park, Seonho Yoo, Kiye Kang, Min-Hee Kang, Youngsok Choi, and Kwonho Hong. "Dynamic Change of R-Loop Implicates in the Regulation of Zygotic Genome Activation in Mouse." International Journal of Molecular Sciences 23, no. 22 (November 18, 2022): 14345. http://dx.doi.org/10.3390/ijms232214345.
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In mice, zygotic genome activation (ZGA) occurs in two steps: minor ZGA at the one–cell stage and major ZGA at the two–cell stage. Regarding the regulation of gene transcription, minor ZGA is known to have unique features, including a transcriptionally permissive state of chromatin and insufficient splicing processes. The molecular characteristics may originate from extremely open chromatin states in the one–cell stage zygotes, yet the precise underlying mechanism has not been well studied. Recently, the R-loop, a triple–stranded nucleic acid structure of the DNA/RNA hybrid, has been implicated in gene transcription and DNA replication. Therefore, in the present study, we examined the changes in R-loop dynamics during mouse zygotic development, and its roles in zygotic transcription or DNA replication. Our analysis revealed that R-loops persist in the genome of metaphase II oocytes and preimplantation embryos from the zygote to the blastocyst stage. In particular, zygotic R-loop levels dynamically change as development proceeds, showing that R-loop levels decrease as pronucleus maturation occurs. Mechanistically, R-loop dynamics are likely linked to ZGA, as inhibition of either DNA replication or transcription at the time of minor ZGA decreases R-loop levels in the pronuclei of zygotes. However, the induction of DNA damage by treatment with anticancer agents, including cisplatin or doxorubicin, does not elicit genome-wide changes in zygotic R-loop levels. Therefore, our study suggests that R-loop formation is mechanistically associated with the regulation of mouse ZGA, especially minor ZGA, by modulating gene transcription and DNA replication.
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Gutierrez, Karina, Werner G. Glanzner, Mariana P. de Macedo, Vitor B. Rissi, Naomi Dicks, Rodrigo C. Bohrer, Hernan Baldassarre, Luis B. Agellon, and Vilceu Bordignon. "Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos." Life 12, no. 2 (January 25, 2022): 171. http://dx.doi.org/10.3390/life12020171.
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CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14–16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts.
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Rengaraj, Deivendran, Sohyoung Won, Jong Won Han, DongAhn Yoo, Heebal Kim, and Jae Yong Han. "Whole-Transcriptome Sequencing-Based Analysis of DAZL and Its Interacting Genes during Germ Cells Specification and Zygotic Genome Activation in Chickens." International Journal of Molecular Sciences 21, no. 21 (October 31, 2020): 8170. http://dx.doi.org/10.3390/ijms21218170.
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The deleted in azoospermia like (DAZL) is required for germ cells development and maintenance. In chickens, the mRNA and protein of DAZL, a representative maternally inherited germ plasm factor, are detected in the germ plasm of oocyte, zygote, and all stages of the intrauterine embryos. However, it is still insufficient to explain the origin and specification process of chicken germ cells, because the stage at which the zygotic transcription of DAZL occurs and the stage at which the maternal DAZL RNA/protein clears have not yet been fully identified. Moreover, a comprehensive understanding of the expression of DAZL interacting genes during the germ cells specification and development and zygotic genome activation (ZGA) is lacking in chickens. In this study, we identified a set of DAZL interacting genes in chickens using in silico prediction method. Then, we analyzed the whole-transcriptome sequencing (WTS)-based expression of DAZL and its interacting genes in the chicken oocyte, zygote, and Eyal-Giladi and Kochav (EGK) stage embryos (EGK.I to EGK.X). In the results, DAZL transcripts are increased in the zygote (onset of transcription), maintained the increased level until EGK.VI, and decreased from EGK.VIII (possible clearance of maternal RNAs). Among the DAZL interacting genes, most of them are increased either at 1st ZGA or 2nd ZGA, indicating their involvement in germ cells specification and development.
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Dresselhaus, Thomas, and Gerd Jürgens. "Comparative Embryogenesis in Angiosperms: Activation and Patterning of Embryonic Cell Lineages." Annual Review of Plant Biology 72, no. 1 (June 17, 2021): 641–76. http://dx.doi.org/10.1146/annurev-arplant-082520-094112.
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Following fertilization in flowering plants (angiosperms), egg and sperm cells unite to form the zygote, which generates an entire new organism through a process called embryogenesis. In this review, we provide a comparative perspective on early zygotic embryogenesis in flowering plants by using the Poaceae maize and rice as monocot grass and crop models as well as Arabidopsis as a eudicot model of the Brassicaceae family. Beginning with the activation of the egg cell, we summarize and discuss the process of maternal-to-zygotic transition in plants, also taking recent work on parthenogenesis and haploid induction into consideration. Aspects like imprinting, which is mainly associated with endosperm development and somatic embryogenesis, are not considered. Controversial findings about the timing of zygotic genome activation as well as maternal versus paternal contribution to zygote and early embryo development are highlighted. The establishment of zygotic polarity, asymmetric division, and apical and basal cell lineages represents another chapter in which we also examine and compare the role of major signaling pathways, cell fate genes, and hormones in early embryogenesis. Except for the model Arabidopsis, little is known about embryopatterning and the establishment of the basic body plan in angiosperms. Using available in situ hybridization, RNA-sequencing, and marker data, we try to compare how and when stem cell niches are established. Finally, evolutionary aspects of plant embryo development are discussed.
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de Frutos, C., R. Laguna-Barraza, P. Bermejo-Alvarez, D. Rizos, and A. Gutierrez-Adan. "91 SPERMATOZOA TELOMERE LENGTH DETERMINES EMBRYONIC TELOMERE LENGTH BEFORE EMBRYONIC GENOME ACTIVATION." Reproduction, Fertility and Development 25, no. 1 (2013): 193. http://dx.doi.org/10.1071/rdv25n1ab91.
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A critical issue for species integrity is the existence of a telomere elongation program during embryogenesis that ensures sufficient telomere reserves in mammalian newborns. Two different mechanisms have been reported to act on telomere elongation during early embryogenesis: first, the telomerase, the ribonucleoprotein that adds telomeric repeats onto the chromosome ends, known to be responsible for the telomere lengthening at the morula-blastocyst transition in mice and bovine; second, in laboratory mice strains, mature oocytes increase the length of their relatively short telomeres between the 1-cell and 2-cell stages by a recombination or ALT-like pathway. In contrast, spermatozoa, the terminally differentiated male gametes, exhibit a very long telomere length (TL). The aim of this study was to clarify the potential role of the spermatozoa TL in the telomere lengthening occurring between oocyte and the 2-cell stage. For this purpose, we used 2 mouse species known to differ greatly in their TL [Mus musculus (hybrid C57CBAF1), long TL, and Mus spretus, short TL]. First, we compared relative TL in sperm samples from 5 age-matched males of each species by quantitative real-time PCR, with the numbers of telomere repeats being normalized, to the amount of DNA present in the sample (based on quantification of the Rn18S gene) by the comparative Ct method. Then, 1- and 2-cell embryos were produced by fertilizing Mus musculus oocytes with either Mus musculus or Mus spretus spermatozoa. The TL analysis in oocytes, zygotes, or 2-cell embryos was carried out by absolute quantification of telomere repeats by qPCR and normalized to the highest Ct observed value. Twenty to thirty samples per stage were analyzed, with each sample consisting in 2 matured oocytes, 2 zygotes, or one 2-cell embryo, to allow comparisons between stages. One-way ANOVA was used for statistical analysis. Mus spretus spermatozoa had significantly shorter telomeres than did Mus musculus (1.0 ± 0.1 v. 9.0 ± 1.5, respectively; P ≤ 0.01). The TL increased after fertilization from oocyte to zygote and 2-cell embryo stages in Mus musculus (1.0 ± 0.1, 1.5 ± 0.1, and 2.4 ± 0.2, respectively; P ≤ 0.01). In contrast, no differences were found in the TLs between the 3 stages in Mus spretus hybrids (oocyte: 1.0 ± 0.1; zygote: 1.0 ± 0.1; and 2-cell embryo: 1.0 ± 0.1), indicating that no elongation occurred after fertilization with spermatozoa with short telomeres. Herein, we demonstrated that before embryonic genome activation occurs, spermatozoa TL determines TL of the early embryo, suggesting that spermatozoon telomeres may act as recombination templates for early telomere lengthening right after syngamia.
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Hamm, Danielle C., and Melissa M. Harrison. "Regulatory principles governing the maternal-to-zygotic transition: insights from Drosophila melanogaster." Open Biology 8, no. 12 (December 2018): 180183. http://dx.doi.org/10.1098/rsob.180183.
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The onset of metazoan development requires that two terminally differentiated germ cells, a sperm and an oocyte, become reprogrammed to the totipotent embryo, which can subsequently give rise to all the cell types of the adult organism. In nearly all animals, maternal gene products regulate the initial events of embryogenesis while the zygotic genome remains transcriptionally silent. Developmental control is then passed from mother to zygote through a process known as the maternal-to-zygotic transition (MZT). The MZT comprises an intimately connected set of molecular events that mediate degradation of maternally deposited mRNAs and transcriptional activation of the zygotic genome. This essential developmental transition is conserved among metazoans but is perhaps best understood in the fruit fly, Drosophila melanogaster . In this article, we will review our understanding of the events that drive the MZT in Drosophila embryos and highlight parallel mechanisms driving this transition in other animals.
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Adenot, P. G., Y. Mercier, J. P. Renard, and E. M. Thompson. "Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos." Development 124, no. 22 (November 15, 1997): 4615–25. http://dx.doi.org/10.1242/dev.124.22.4615.
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In the mouse embryo, transcriptional activation begins during S/G2 phase of the first cell cycle when paternal and maternal chromatin are still in separate nuclear entities within the same cytoplasm. At this time, the male pronucleus exhibits greater transcriptional activity than the female pronucleus. Since acetylation of histones in the nucleosome octamer exerts a regulatory influence on gene expression, we investigated changes in histone acetylation during the remodeling of paternal and maternal chromatin from sperm entry through to minor genome activation and mitosis. We found (1) neither mature sperm nor metaphase II maternal chromatin stained for hyperacetylated histone H4; (2) immediately following fertilization, hyperacetylated H4 was associated with paternal but not maternal chromatin while, in parthenogenetically activated oocytes, maternal chromatin became hyperacetylated; (3) in zygotes, differential levels and patterns of hyperacetylated H4 between male and female pronuclei persisted throughout most of G1 with histone deacetylases and acetyltransferases already active at this time; (4) when transcriptional differences are observed in S/G2, male and female pronuclei have equivalent levels of H4 hyperacetylation and DNA replication was not required to attain this equivalence and (5) in contrast to the lack of H4 hyperacetylation on gametic chromatin, chromosomes at the first mitosis showed distinct banding patterns of H4 hyperacetylation. These results suggest that sperm chromatin initially out-competes maternal chromatin for the pool of hyperacetylated H4 in the oocyte, that hyperacetylated H4 participates in the process of histone-protamine exchange in the zygote, and that differences in H4 acetylation in male and female pronuclei during G1 are translated across DNA replication to transcriptional differences in S/G2. Prior to fertilization, neither paternal nor maternal chromatin show memory of H4 hyperacetylation patterns but, by the end of the first cell cycle, before major zygotic genome activation at the 2-cell stage, chromosomes already show hyperacetylated H4 banding patterns.
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Goszczynski, D. E., P. Tinetti, Y. H. Choi, K. Hinrichs, and P. J. Ross. "59 Genome activation in intracytoplasmic sperm injection-derived horse embryos." Reproduction, Fertility and Development 32, no. 2 (2020): 155. http://dx.doi.org/10.1071/rdv32n2ab59.
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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.
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Lepikhov, K., F. Yang, C. Wrenzycki, V. Zakhartchenko, H. Niemann, E. Wolf, and J. Walter. "131 DYNAMICS OF HISTONE H3 METHYLATION AT POSITIONS K4 AND K9 IN MOUSE, RABBIT, AND BOVINE PRE-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 18, no. 2 (2006): 174. http://dx.doi.org/10.1071/rdv18n2ab131.
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In mammals, upon the penetration of sperm into the oocyte, the paternal genome undergoes dramatic epigenetic changes. Protamin packaging of DNA is replaced by histones that acquire specific modifications. In mouse zygotes, paternal DNA gets rapidly demethylated by an active mechanism. In bovine zygotes the methylation from paternal DNA is erased only partially, and in rabbit zygotes it persists at the initial level. To understand whether these reprogramming differences are also reflected in histone modifications, we examined the dynamic changes of histone H3 methylation at positions K4 and K9 in mouse, bovine, and rabbit zygotes and in preimplantation embryos using an immunofluorescence staining procedure (Lepikhov and Walter 2004 BMC Dev. Biol. 4, 12). In zygotes, maternal chromatin contains both types of histone H3 methylation. After fertilization protamines in sperm are very quickly replaced by histones. After the formation of nucleosomes, histone H3 acquires methylation at position K4 in a stepwise manner: first as mono-methylated form and later as tri-methylated. In the late zygote, both paternal and maternal pronuclei show equal levels of histone H3 methylation at position K4. Regardless of the differences in DNA reprogramming in these 3 species, H3/K9 di-methylation is not detected on paternal genomes and is only associated with maternal genomes. During the subsequent cleavage stages, H3/K9 di-methylation decreases gradually and becomes hardly detectable in 4-cell bovine and rabbit embryos. In mouse embryos, it is detectable through all the stages. Bovine embryos reacquire this type of modification at the 8-16 cell stage, and it remains at the very low levels in rabbit, embryos until the blastocyst stage. In conclusion, mouse, rabbit and bovine zygotes show similar patterns of H3/K4triMe and H3/K9diMe distribution despite the difference in paternal DNA demethylation. The dynamics of H3/K9diMe distribution patterns in cleavage stage embryos from all embryos do not correlate with embryonic genomic activation events.
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Whitworth, K. M., S. L. Murphy, J. A. Benne, L. D. Spate, E. Walters, R. Hickey, S. L. Nyberg, K. D. Wells, and R. S. Prather. "25 GENOME EDITING OF SOMATIC CELL NUCLEAR TRANSFER DERIVED ZYGOTES BY CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 GUIDE RNA INJECTION." Reproduction, Fertility and Development 28, no. 2 (2016): 142. http://dx.doi.org/10.1071/rdv28n2ab25.
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Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have greatly improved the efficiency of genome editing in pigs. However, in some cases, genetically modified pig models need an additional modification to improve their application. The objective of this experiment was to determine whether a combination of somatic cell NT (SCNT) by using a previously modified donor cell line and subsequent zygote injection with CRISPR/Cas9 guide RNA to target a second gene would result in embryos and offspring successfully containing both modifications. Fibroblast cell lines were collected from fumarylacetoacetate hydrolase deficient (FAH–/–) fetuses and used as the donor cell line. Somatic cell NT was performed by standard technique. A CRISPR guide RNA specific for recombination activating gene 2 (RAG2) was designed and in vitro transcribed from a synthesised gBlock (IDT) containing a T7 promoter sequence, the CRISPR Guide RNA (20 bp), and 85 bp of tracer RNA. The gBlock was PCR amplified with Q5 polymerase (NEB, Ipswich, MA, USA) and in vitro transcribed with the MEGAshortscript™ T7 Transcription Kit (Life Technologies, Grand Island, NY, USA). Guide RNA (20 ng μL–1) and polyadenylated Cas9 (20 ng μL–1, Sigma, St. Louis, MO, USA) were co-injected into the cytoplasm of SCNT zygotes at 14 to 16 h after fusion and activation. Injected SCNT were then cultured in vitro in PZM3 + 1.69 mM arginine medium (MU1) to Day 5. Three embryo transfers were performed surgically into recipient gilts on Day 4 or 5 of oestrus (50, 62, or 70 embryos per pig) to evaluate in vivo development. The remaining embryos were cultured in MU1 to Day 7 and analysed for the presence of modifications to the RAG2 gene. Embryos were classified as modified if they contained an INDEL as measured by both gel electrophoresis and DNA sequencing of PCR amplicons spanning the targeted exon. The RAG2 modification rate was 83.3% (n = 6), of which 50% (n = 3) of the embryos contained biallelic modifications. All control embryos contained a wild-type RAG2 gene (n = 5). Embryo transfer resulted in a 33.3% pregnancy rate (1/3). The combination of SCNT and CRISPR/Cas9 zygote injection can be a highly efficient tool to successfully create pig embryos with an additional modification. This additional technique further improves the usefulness of already created genetically modified pig models. This study was funded by the National Institutes of Health via U42 OD011140.
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Ao, Asangla, Robert P. Erickson, Robert M. L. Winston, and Alan H. Handysude. "Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage." Zygote 2, no. 4 (November 1994): 281–87. http://dx.doi.org/10.1017/s0967199400002100.
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SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.
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Fraser, Rupsha, and Chih-Jen Lin. "Epigenetic reprogramming of the zygote in mice and men: on your marks, get set, go!" Reproduction 152, no. 6 (December 2016): R211—R222. http://dx.doi.org/10.1530/rep-16-0376.
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Gametogenesis (spermatogenesis and oogenesis) is accompanied by the acquisition of gender-specific epigenetic marks, such as DNA methylation, histone modifications and regulation by small RNAs, to form highly differentiated, but transcriptionally silent cell-types in preparation for fertilisation. Upon fertilisation, extensive global epigenetic reprogramming takes place to remove the previously acquired epigenetic marks and produce totipotent zygotic states. It is the aim of this review to delineate the cellular and molecular events involved in maternal, paternal and zygotic epigenetic reprogramming from the time of gametogenesis, through fertilisation, to the initiation of zygotic genome activation for preimplantation embryonic development. Recent studies have begun to uncover the indispensable functions of epigenetic players during gametogenesis, fertilisation and preimplantation embryo development, and a more comprehensive understanding of these early events will be informative for increasing pregnancy success rates, adding particular value to assisted fertility programmes.
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Liu, Tiancheng, Lin Yu, Guohui Ding, Zhen Wang, Lei Liu, Hong Li, and Yixue Li. "Gene Coexpression and Evolutionary Conservation Analysis of the Human Preimplantation Embryos." BioMed Research International 2015 (2015): 1–11. http://dx.doi.org/10.1155/2015/316735.
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Evolutionary developmental biology (EVO-DEVO) tries to decode evolutionary constraints on the stages of embryonic development. Two models—the “funnel-like” model and the “hourglass” model—have been proposed by investigators to illustrate the fluctuation of selective pressure on these stages. However, selective indices of stages corresponding to mammalian preimplantation embryonic development (PED) were undetected in previous studies. Based on single cell RNA sequencing of stages during human PED, we used coexpression method to identify gene modules activated in each of these stages. Through measuring the evolutionary indices of gene modules belonging to each stage, we observed change pattern of selective constraints on PED for the first time. The selective pressure decreases from the zygote stage to the 4-cell stage and increases at the 8-cell stage and then decreases again from 8-cell stage to the late blastocyst stages. Previous EVO-DEVO studies concerning the whole embryo development neglected the fluctuation of selective pressure in these earlier stages, and the fluctuation was potentially correlated with events of earlier stages, such as zygote genome activation (ZGA). Such oscillation in an earlier stage would further affect models of the evolutionary constraints on whole embryo development. Therefore, these earlier stages should be measured intensively in future EVO-DEVO studies.
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Schultz, GA, A. Hogan, AJ Watson, RM Smith, and S. Heyner. "Insulin, insulin-like growth factors and glucose transporters: temporal patterns of gene expression in early murine and bovine embryos." Reproduction, Fertility and Development 4, no. 4 (1992): 361. http://dx.doi.org/10.1071/rd9920361.
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mRNA phenotyping by the reverse transcription-polymerase chain reaction (RT-PCR) method was used to compare the patterns of expression of insulin and insulin-like growth factor (IGF) ligand and receptor genes in preimplantation bovine embryos with those established previously for preimplantation murine embryos. In the early bovine embryo, transcripts for IGF-I, IGF-II and mRNAs encoding receptors for insulin, IGF-I and IGF-II were all detectable at all embryo stages from the 1-cell zygote to the blastocyst. In the mouse, IGF-II ligand and receptor mRNAs were not expressed until the 2-cell stage, and the insulin and IGF-I receptor mRNAs were not detectable until the 8-cell stage. Since transcriptional activation of the embryonic genome occurs at the 8- to 16-cell stage in the bovine embryo and at the 2-cell stage in the murine embryo, it is suggested that these transcripts are products of both the maternal and embryonic genomes in the bovine embryo whereas in the mouse they are present only after activation of the embryonic genome. Transcripts for insulin were not detected in preimplantation embryos of either species. Colloidal-gold immunocytochemistry with antibodies directed against the insulin receptor, IGF-I receptor and IGF-I ligand has confirmed the presence of these molecules in bovine blastocysts. RT-PCR and indirect immunofluorescence procedures demonstrated that the glucose transporter (GLUT) isoform 1 is present in murine embryos from the oocyte to blastocyst stage whereas GLUT 2 expression begins at the 8-cell stage.
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Xu, Jiawei, Yimin Shu, Guidong Yao, Yu Zhang, Wenbin Niu, Yile Zhang, Xueshan Ma, et al. "Parental methylome reprogramming in human uniparental blastocysts reveals germline memory transition." Genome Research 31, no. 9 (July 30, 2021): 1519–30. http://dx.doi.org/10.1101/gr.273318.120.
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Uniparental embryos derived from only the mother (gynogenetic [GG]) or the father (androgenetic [AG]) are unique models for studying genomic imprinting and parental contributions to embryonic development. Human parthenogenetic embryos can be obtained following artificial activation of unfertilized oocytes, but the production of AG embryos by injection of two sperm into one denucleated oocyte leads to an extra centriole, resulting in multipolar spindles, abnormal cell division, and developmental defects. Here, we improved androgenote production by transferring the male pronucleus from one zygote into another haploid androgenote to prevent extra centrioles and successfully generated human diploid AG embryos capable of developing into blastocysts with an identifiable inner cell mass (ICM) and trophectoderm (TE). The GG embryos were also generated. The zygotic genome was successfully activated in both the AG and GG embryos. DNA methylome analysis showed that the GG blastocysts partially retain the oocyte transcription-dependent methylation pattern, whereas the AG blastocyst methylome showed more extensive demethylation. The methylation states of most known imprinted differentially methylated regions (DMRs) were recapitulated in the AG and GG blastocysts. Novel candidate imprinted DMRs were also identified. The production of uniparental human embryos followed by transcriptome and methylome analysis is valuable for identifying parental contributions and epigenome memory transitions during early human development.
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Kageyama, S., W. Gunji, M. Nakasato, Y. Murakami, M. Nagata, and F. Aoki. "Analysis of transcription factor expression during oogenesis and preimplantation development in mice." Zygote 15, no. 2 (May 2007): 117–28. http://dx.doi.org/10.1017/s096719940700411x.
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SummaryThe transition from a differentiated germ cell into a totipotent zygote during oogenesis and preimplantation development is critical to the creation of a new organism. During this period, cell characteristics change dynamically, suggesting that a global alteration of gene expression patterns occurs, which is regulated by global changes in various epigenetic factors. Among these, transcription factors (TFs) are essential in the direct regulation of transcription and also play important roles in determining cell characteristics. However, no comprehensive analysis of TFs from germ cells to embryos had been undertaken. We used mRNA amplification systems and microarrays to conduct a genomewide analysis of TFs at various stages of oogenesis and preimplantation development. The greatest alteration in TFs occurred between the 1- and 2-cell stages, at which time zygotic genome activation (ZGA) occurs. Our analysis of TFs classified by structure and function revealed several specific patterns of change. Basic transcription factors, which are the general components of transcription, increased transiently at the 2-cell stage, while homeodomain (HD) TFs were expressed specifically in the oocyte. TFs containing the Rel homology region (RHR) and Ets domains were expressed at a high level in 2-cell and blastocyst embryos. Thus, the global TF dynamics that occur during oogenesis and preimplantation development seem to regulate the transition from germ-cell-type to embryo-type gene expression.
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Höffmann, K., H. Niemann, K. G. Hadeler, D. Herrmann, and C. Wrenzycki. "247 MESSENGER RNA EXPRESSION PATTERNS OF DNA AND HISTONE METHYLTRANSFERASES IN PREIMPLANTATION DEVELOPMENT OF IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS." Reproduction, Fertility and Development 18, no. 2 (2006): 231. http://dx.doi.org/10.1071/rdv18n2ab247.
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The effects of in vitro production (IVP) and/or somatic nuclear transfer on mRNA expression patterns have mostly been determined in morulae and blastocysts, i.e. after embryonic genome activation. Comparative data regarding mRNA expression patterns throughout the oviductal phase of pre-implantation development are scarce. Here we studied mRNA expression for genes related to DNA methylation and modification of histones which account for the major epigenetic reprogramming during development. Pertubated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases associated with assisted reproduction technologies. The objective of the present study was to compare mRNA expression of DNA methyltransferases Dnmt1, -3a, and -3b and histone methyltransferases SUV39-h1 and G9a between in vivo-derived bovine embryos and their IVP counterparts using a semiquantitative RT-PCR assay (Wrenzycki et al. 2002 Biol. Reprod. 66, 127-134) employing two embryos for each assay. In vivo-derived embryos were collected from 28 superovulated heifers by endoscopic flushing of oviducts (zygotes to 8-cell stages) (Besenfelder et al. 2001 Theriogenology 55, 837-845) or by uterine flushing (16-cell stages to blastocysts). Endoscopic flushing at different time points after AI (Days 1, 1.5, 2, 3, 4, and 4.5) yielded 31 zygotes; 15 two-cell, 5 three-cell, 13 four-cell, 1 five-cell, 2 six-cell, and 11 eight-cell embryos; 4 degenerated embryos; and 18 unfertilized ova. The recovery rate (corpora lutea counted per recovered embryos) was 58% and 62% for the endoscopic and uterine flushing, respectively. Differences in the relative abundance of each gene transcript between groups were tested using ANOVA with the main effects being origin (in vivo/in vitro) and developmental stage (zygote to blastocyst) and their interactions followed by multiple pairwise comparisons using a Tukey test (P < 0.05). Origin of embryos affected the relative abundance of transcripts for Dnmt1, Dnmt3a, and SUV39-h1, and developmental stage affected the relative abundance of transcripts for Dnmt1, -3a, -3b, SUV39-h1, and G9a. No interactive effects were observed for origin and developmental stage in the relative abundance of all transcripts. The relative abundance of Dnmt1 transcripts differed significantly between in vivo- and in vitro-produced morulae and blastocysts. For Dnmt3a, mRNA differences were determined between in vivo- and in vitro-produced 10-16-cell stages and morulae. Suv39-h1 transcripts differed significantly between in vivo- and in vitro-derived zygotes, 2-cell embryos, 8-cell embryos, 10-16-cell embryos, and blastocysts. The results suggest that IVP alters mRNA expression of genes related to epigenetic modifications very early in development, even before the embryonic genome has been activated.
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Christou-Kent, Marie, Magali Dhellemmes, Emeline Lambert, Pierre F. Ray, and Christophe Arnoult. "Diversity of RNA-Binding Proteins Modulating Post-Transcriptional Regulation of Protein Expression in the Maturing Mammalian Oocyte." Cells 9, no. 3 (March 9, 2020): 662. http://dx.doi.org/10.3390/cells9030662.
Full textAbstract:
The oocyte faces a particular challenge in terms of gene regulation. When oocytes resume meiosis at the end of the growth phase and prior to ovulation, the condensed chromatin state prevents the transcription of genes as they are required. Transcription is effectively silenced from the late germinal vesicle (GV) stage until embryonic genome activation (EGA) following fertilisation. Therefore, during its growth, the oocyte must produce the mRNA transcripts needed to fulfil its protein requirements during the active period of meiotic completion, fertilisation, and the maternal-to zygote-transition (MZT). After meiotic resumption, gene expression control can be said to be transferred from the nucleus to the cytoplasm, from transcriptional regulation to translational regulation. Maternal RNA-binding proteins (RBPs) are the mediators of translational regulation and their role in oocyte maturation and early embryo development is vital. Understanding these mechanisms will provide invaluable insight into the oocyte’s requirements for developmental competence, with important implications for the diagnosis and treatment of certain types of infertility. Here, we give an overview of post-transcriptional regulation in the oocyte, emphasising the current knowledge of mammalian RBP mechanisms, and develop the roles of these mechanisms in the timely activation and elimination of maternal transcripts.
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Farrell, K., K. Uh, and K. Lee. "45 Expression patterns of PRDM family genes in porcine pre-implantation embryos." Reproduction, Fertility and Development 32, no. 2 (2020): 148. http://dx.doi.org/10.1071/rdv32n2ab45.
Full textAbstract:
Establishing proper levels of pluripotency is essential for normal development. The genome of gametes is remodelled upon fertilisation and pluripotency-related genes are expressed in blastocysts. Multiple pluripotency-related genes are involved in the well-orchestrated process; however, detailed mechanistic actions remain elusive. The PRDM family genes are reported to be closely related to the pluripotency. A previous report noted that PRDM14 plays an important role in the maintenance of pluripotency in human embryonic stem cells (ESCs) and potentially murine ESCs; loss of PRDM14 was found to cause abnormalities in genome-wide epigenetic status. Similarly, PRDM15 was found to be a key regulator of pluripotency in mouse ESCs. Structural similarities among the PRDM family suggest that other PRDM family genes may help to establish and maintain pluripotency in embryos. Unfortunately, little is known about the expression profile of PRDM family in porcine embryos. To expand our understanding of the role of PRDM family in porcine embryos, expression patterns of PRDM gene family were investigated using reverse transcription quantitative (RTq)-PCR. Candidate PRDM family genes were selected based on previous RNA-Seq data in porcine oocytes/embryos. To conduct this study, germinal vesicle (GV), MII, zygote, 4-cell, and blastocyst samples were collected. Complementary DNA synthesised from the samples was used for RT-qPCR to analyse the expression pattern of selected PRDM family genes: PRDM2, PRDM4, PRDM6, PRDM14, and PRDM15. The expression of target genes was normalized to the YWHAG level, an internal control. Then, GV stage was used as a control for ΔΔCT analysis. Two technical replications and three biological replications were performed. Analysis of variance was used for statistical analysis and P-values<0.05 were considered significant. There was a significant decrease in PRDM2 expression in 4-cell and blastocyst, PRDM4 expression in 4-cell, and PRDM6 in all stages (MII, zygote, 4-cell, and blastocyst), compared with the GV stage. Because zygotic genome activation occurs at the 4-cell stage in the pig, the significant decrease in gene expression (PRDM2, PRDM4, and PRDM6) indicates they may be maternally originated and involved in the reprogramming process following fertilisation. On the other hand, there was a significant increase in PRDM15 expression in blastocysts and the PRDM14 transcript was only detected in blastocysts in all three biological replicates, suggesting that the genes are most likely involved in pluripotency maintenance, as was found in previous human studies. These results indicate that PRDM family genes are differentially expressed during early embryo development in pigs and may play a role in maintenance of pluripotency. For further study, we intend to evaluate the role of PRDM family genes during early embryo development in pigs.
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Bebbere, Daniela, Luisa Bogliolo, Federica Ariu, Stefano Fois, Giovanni Giuseppe Leoni, Silvia Tore, Sara Succu, Fiammetta Berlinguer, Salvatore Naitana, and Sergio Ledda. "Expression pattern of zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes in ovine oocytes and in vitro-produced preimplantation embryos." Reproduction, Fertility and Development 20, no. 8 (2008): 908. http://dx.doi.org/10.1071/rd08095.
Full textAbstract:
The expression patterns of four maternal effect genes (MEG), namely zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), were determined in ovine oocytes and in vitro-produced preimplantation embryos. The existence of ZAR1 and MATER in ovine species has not been reported previously. Reverse transcription–polymerase chain reaction was performed on germinal vesicle and IVM MII oocytes, as well as in in vitro fertilised and cultured two-, four-, eight- and 12/16-cell embryos, morulae and blastocysts. Quantification of gene expression by real-time polymerase chain reaction showed the highest abundance of all transcripts analysed in the immature oocyte. During the following stages of preimplantation development, the mRNAs examined exhibited different patterns of expression, but often significant decreases were observed during maturation and maternal–embryonic transition. The transcription of the four genes did not resume with activation of the genome.
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Zhang, K., and H. Wang. "61 Expression Profiles and Functional Roles of H3.3 and HIRA in Bovine Early Embryos." Reproduction, Fertility and Development 30, no. 1 (2018): 169. http://dx.doi.org/10.1071/rdv30n1ab61.
Full textAbstract:
Early embryo death is one major reason for poor reproductive efficiency in dairy cows. In particular, ~20 to 50% of high-producing cows are subject to pregnancy loss during the first week of gestation, indicating the importance of embryonic development from fertilization to the blastocyst stage. To highlight this importance, multiple critical molecular and developmental events, including zygote reprogramming, maternal RNA decay, and embryonic genome activation, occur during bovine pre-implantation development. However, the molecular mechanisms of these events have yet to be defined. H3.3 is a histone H3 variant that encoded by 2 genes, namely, H3F3A and H3F3B. It is generally believed that H3.3 is closely related to active transcribed genes. Of interest, H3.3 required for establishing proper chromatin structure during mouse oogenesis. Immediately following fertilization, H3.3 is incorporated to parental chromatins and essential for blastocyst formation in mice. HIRA is a chaperone for H3.3 deposition and indispensable for zygote development. Previously, our results showed that H3.3 is needed for bovine early embryonic development. Herein, experiments were designed to determine the mechanisms of functional requirement of H3.3 in bovine early embryos. Slaughterhouse-derived cumulus–oocyte complexes (COC) were matured in vitro and IVF was performed. To knock down genes of interest, small interfering (si)RNAs were delivered into zygotes via microinjection. The qPCR results showed that H3F3A mRNA level is stable, whereas H3F3B and HIRA mRNA are dynamic during early embryonic development (4 replicates). The mRNA abundance of H3F3B is significantly higher than that of H3F3A (4 replicates; P < 0.05), which is also found in mouse and human. Immunostaining results revealed a stage-specific pattern for the localization of H3.3 in bovine early embryos, and the H3.3 signal was not different between paternal and maternal pronuclei in zygotes, which was different from the pattern in mice. The siRNA-mediated silencing of H3.3 dramatically reduces the expression of CTGF (a putative trophectoderm marker) in bovine blastocysts (3 replicates; P < 0.05). Furthermore, we found that the signal intensity of dimethylation of histone H3 lysine 36 (H3K36me2) and linker histone H1 decreases in H3.3-ablated embryos, which is similar to CHD1 knockdown (3 replicates; P < 0.05). However, no difference was found for the intensity of trimethylation of histone H3 lysine 4, dimethylation of histone H3 lysine 9 (H3K9me2) and splicing factor 3 B1 (SF3B1). We also found that HIRA deletion does not affect bovine early embryonic development. Taken together, the results described herein suggest that H3.3 is required for proper epigenetic modifications and H1 deposition during bovine early embryonic development. This project was supported by National Natural Science Foundation of China grant (No. 31672416) and the Fundamental Research Funds for the Central Universities.
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Jin, X. L., and C. O'Neill. "The regulation of the expression and activation of the essential ATF1 transcription factor in the mouse preimplantation embryo." REPRODUCTION 148, no. 2 (August 2014): 147–57. http://dx.doi.org/10.1530/rep-13-0535.
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The co-expression of the CREB and ATF1 transcription factors is required for the development of preimplantation embryos. Embryotropin-mediated, calcium/calmodulin-dependent signalling activates CREB-induced transcription in the two-cell embryo, but the regulation of ATF1 in the embryo is not known. This study demonstrates that ATF1 begins to accumulate within both pronuclei of the mouse zygote by 20 h post-human chorionic gonadotrophin. This did not require new transcription (not blocked by α-amanitin), but was dependent upon protein synthesis (blocked by puromycin) and the activity of P38 MAP kinase. ATF1 becomes an active transcription factor upon being phosphorylated. A marked accumulation of phosphorylated ATF1 was evident in two-cell embryos and this persisted in subsequent stages of development. This phosphorylation was enhanced by the actions of autocrine embryotropic mediators (including Paf) and required the mutual actions of P38 MAP kinase and calmodulin-dependent pathways for maximum levels of phosphorylation. The combined inhibition of these two pathways blocked embryonic genome activation (EGA) and caused embryos to enter a developmental block at the two-cell stage. The members of the CREB family of transcription factors can generate one of the most diverse transcriptomes of any transcription factor. The demonstration of the presence of activated CREB and ATF1 within the embryonic nucleus at the time of EGA places these transcription factors as priority targets as key regulators of EGA.
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Xu, Tengteng, Chengxue Liu, Mengya Zhang, Xin Wang, Yelian Yan, Qiuchen Liu, Yangyang Ma, et al. "Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation." Animals 12, no. 5 (February 28, 2022): 610. http://dx.doi.org/10.3390/ani12050610.
Full textAbstract:
Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (α-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation.
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Madeja, Zofia E., Piotr Pawlak, and Anna Piliszek. "Beyond the mouse: non-rodent animal models for study of early mammalian development and biomedical research." International Journal of Developmental Biology 63, no. 3-4-5 (2019): 187–201. http://dx.doi.org/10.1387/ijdb.180414ap.
Full textAbstract:
The preimplantation development of mammals generally follows the same plan. It starts with the formation of a totipotent zygote, and through consecutive cleavage divisions and differentiation events leads to blastocyst formation. However, the intervening events may differ between species. The regulation of these processes has been extensively studied in the mouse, which displays some unique features among eutherian mammals. Farm animals such as pigs, cattle, sheep and rabbits share several similarities with one another, and with the human developmental plan. These include the timing of epigenetic reprogramming, the moment of embryonic genome activation and the developmental time-frame. Recently, efficient techniques for genetic modification have been established for large domestic animals. Genome sequences and gene manipulation tools are now available for cattle, pigs, sheep and goats, and a larger number of genetically engineered livestock is now accessible for biomedical research. Yet, these animals still make up less than 0.5% of animals in research, mainly due to our inadequate knowledge of the processes responsible for pluripotency maintenance (to date no stable naïve embryonic stem cell lines have been established) and early development. In this review, we highlight our present knowledge of the key preimplantation events in the 3 non-rodent species which present the highest potential for biomedical research related to early embryonic development: cattle, which offer an excellent model to study human in vitro embryo development, pigs which emerge as models to study the long-term effects of gene-based therapies and rabbits, which in many aspects of embryology resemble the human.
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Lepikhov, K., V. Zakhartchenko, F. Yang, C. Wrenzycki, E. Wolf, and J. Walter. "110 COMPARATIVE ANALYSIS OF DNA AND HISTONE H3 METHYLATION REPROGRAMMING IN MOUSE, BOVINE, AND RABBIT ZYGOTES." Reproduction, Fertility and Development 20, no. 1 (2008): 135. http://dx.doi.org/10.1071/rdv20n1ab110.
Full textAbstract:
Active demethylation of paternal DNA in zygotes has been documented for many mammalian species, including mouse (Mayer et al. 2000 Nature 403, 501–505), rat (Zaitseva et al. 2007 Mol. Reprod. Dev. 74, 1255–1261), pig (Fulka et al. 2006 Histochem. Cell. Biol. 126, 213–217), Homo sapiens (Fulka et al. 2004 Reproduction 128, 703–708), and cow (Beaujean et al. 2004 Curr. Biol. 14, 266–267). The generality of this concept has been challenged by reports stating that there is no detectable genome-wide paternal DNA demethylation in rabbit (Shi et al. 2004 Biol. Reprod. 71, 340–347) and ovine (Beaujean et al. 2004 Curr. Biol. 14, 266–267), whereas others report partial evidence for the opposite (Zhang et al. 2005 Mol. Reprod. Dev. 72, 530–533). In order to compare the fate of DNA and histone H3 methylation (H3/K4triMe and H3/K9diMe), we performed studies on mouse, bovine, and rabbit zygotes using specific antibodies in an indirect immunofluorescence approach. We observed a similar distribution of these epigenetic modifications in maternal and paternal pronuclei of the zygote, indicating that the overall epigenetic reprogramming activities — including the DNA demethylation of the paternal pronucleus — are conserved between mammalian species. For all 3 species we found a clear asymmetry in mouse monoclonal antimethylcytosine (MeC) antibody signal intensities between paternal and maternal pronuclei at advanced stages, which suggests the presence of paternal DNA demethylation. To obtain additional evidence for the presence of DNA demethylation activity in rabbit zygotes, we examined 1-cell embryos after somatic cell nuclear transfer (SCNT) using rabbit fetal fibroblasts as nuclear donors, and we found a strong signal reduction in SCNT embryos 4 h after activation. For the majority of SCNT 1-cell embryos, we estimated at least a 4- to 6-fold decrease of MeC signal. Along with the similarities of DNA demethylation kinetics in rabbit, mouse, and bovine zygotes, we found a conserved distribution of H3/K4triMe and H3/K9diMe signals on maternal and paternal chromatin. In all 3 species, H3/K4triMe, which predominantly demarcate open chromatin, is present in both pronuclear chromatin, whereas H3/K9diMe, a hallmark of condensed chromatin, is strongly enriched in maternal pronuclear chromatin and undetectable in paternal chromatin. Furthermore, we observed a similar asymmetric compartmentalization of paternal and maternal chromosomes in early 2-cell rabbit and bovine embryos as monitored by H3/K9diMe, which marks maternally derived chromosomes. In summary, our data suggest that mechanisms of epigenetic reprogramming are conserved in mammalian species both on the level of DNA and chromatin methylation.
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Datta, Tirtha K., Sandeep K. Rajput, Gabbine Wee, KyungBon Lee, Joseph K. Folger, and George W. Smith. "Requirement of the transcription factor USF1 in bovine oocyte and early embryonic development." REPRODUCTION 149, no. 2 (February 2015): 203–12. http://dx.doi.org/10.1530/rep-14-0445.
Full textAbstract:
Upstream stimulating factor 1 (USF1) is a basic helix–loop–helix transcription factor that specifically binds to E-box DNA motifs, knowncis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated thatUSF1mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8–16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown ofUSF1in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion ofUSF1in presumptive zygote stage embryos demonstrated thatUSF1is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation ofJY1, GDF9, andFSTgenes associated with oocyte competence.
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Ménézo, Yves, Brian Dale, and Marc Cohen. "DNA damage and repair in human oocytes and embryos: a review." Zygote 18, no. 4 (July 21, 2010): 357–65. http://dx.doi.org/10.1017/s0967199410000286.
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SummaryThe genome of all cells is protected at all times by mechanisms collectively known as DNA repair activity (DRA). Such activity is particularly important at the beginning of human life, i.e. at fertilization, immediately after and at the very onset of embryonic development. DRA in early development is, by definition, of maternal origin: the transcripts stored during maturation, need to control the integrity of chromatin, at least until the maternal/zygotic transition at the 4- to 8-cell stage in the human embryo. Tolerance towards DNA damage must be low during this critical stage of development. The majority of DNA damage is due to either apoptosis or reactive oxygen species (ROS). Apoptosis, abortive or not, is a common feature in human sperm, especially in oligoasthenospermic patients and FAS ligand has been reported on the surface of human spermatozoa. The susceptibility of human sperm to DNA damage is well documented, particularly the negative effect of ROS (Kodama et al., 1997; Lopes et al., 1998a, b) and DNA modifying agents (Zenzes et al., 1999; Badouard et al., 2007). DNA damage in sperm is one of the major causes of male infertility and is of much concern in relation to the paternal transmission of mutations and cancer (Zenzes, 2000; Aitken et al., 2003; Fernández-Gonzalez, 2008). It is now clear that DNA damaged spermatozoa are able to reach the fertilization site in vivo (Zenzes et al., 1999), fertilize oocytes and generate early embryos both in vivo and in vitro. The effect of ROS on human oocytes is not as easy to study or quantify. It is a common consensus that the maternal genome is relatively well protected while in the maturing follicle; however damage may occur during the long quiescent period before meiotic re-activation (Zenzes et al., 1998). In fact, during the final stages of follicular growth, the oocyte may be susceptible to damage by ROS. With regards to the embryo there is active protection against ROS in the surrounding environment i.e. in follicular and tubal fluid (El Mouatassim et al., 2000; Guerin et al., 2001). DNA repair activity in the zygote is mandatory in order to avoid mutation in the germ line (Derijck et al., 2008). In this review we focus on the expression of mRNAs that regulate DNA repair capacity in the human oocyte and the mechanisms that protect the embryo against de novo damage.
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Kues, W. A., K. Iqbal, B. Barg-Kues, and H. Niemann. "305 REPROGRAMMING EVENTS IN EARLY BOVINE AND MURINE EMBRYOS ARE MIRRORED BY PLASMID-ENCODED MARKER CONSTRUCTS." Reproduction, Fertility and Development 21, no. 1 (2009): 249. http://dx.doi.org/10.1071/rdv21n1ab305.
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Episomal plasmids have emerged as useful tools to achieve stable transgenesis in mammalian cell cultures. Here, the suitability of scaffold/matrix attachment region (S/MAR) carrying episomal plasmids and conventional plasmids for the generation of transgenic murine and bovine embryos was assessed. Bovine zygotes were produced from slaughterhouse ovaries, and murine zygotes were isolated from superovulated and mated NMRI females. Zygote stages were microinjected with approximately 10 pl of plasmid solution. The S/MAR encoding plasmids pEPI or minicircle preparations (gift of J. Bode, Braunschweig, Germany), devoid of most of the plasmid backbone, were used as episomal plasmids. Both plasmids carry an enhanced green fluorescent protein (EGFP) gene driven by the cytomegalovirus promoter (CMV). The plasmids peGFP (CMV-eGFP), pdsRED encoding red fluorescent protein (CMV-RFP), pOct4-GFP (germ line-specific Oct-4 promoter-EGFP), and pgAChR-GFP (muscle-specific γAChR promoter) were used as conventional plasmids. To study the effects of DNA methylation at cytosine/guanine dinucleotids (CpG), plasmid DNA was treated with CpG-methylase in the presence of S-adenosyl-methionin, and in some experiments, completeness of DNA methylation was verified by methylation-sensitive restriction endonucleases. Embryos were analyzed during in vitro culture up to blastocyst stage by fluorescence microscopy, and selected stages were harvested for RT-PCR analysis or DNA recovery. Microinjection of circular plasmids with ubiquitous CMV (n = 505) or germ line-specific Oct-4 promoter (n = 176) driven transcription in bovine zygotes resulted in 159 and 44 blastocysts, of which 94 and 27 showed expression of EGFP. Microinjection of bovine zyotes (n = 179) with S/MAR plasmids yielded a total of 18 blastocysts of which 12 were green fluorescent protein-positive. On average, >50% of the blastocysts were EGFP-positive, irrespective of whether S/MAR carrying episomal plasmids or conventional plasmids had been injected. In contrast, injection of the γAChR (muscle-specific) driven construct did not give rise to EGFP expression (n = 20), suggesting that promoter specificity was maintained. Injection of murine zygotes (n = 126) with CMV or Oct-4 promoter constructs was less successful, about 10 to 20% of the obtained blastocysts expressed EGFP. In the case of unmethylated pOct4-GFP plasmid, the onset of EGFP expression was found to coincide with the time point of major embryonic genome activation [i.e. late 1-cell stage in murine (n = 25) and 4- to 8-cell stages in bovine (n = 75) embryos]. In contrast, injection of CpG-methylated plasmids (murine n = 33; bovine n = 101) delayed the onset of EGFP expression for a further 30 to 40 h. Recovery of plasmid sequences from blastocyst stages and bisulfite sequencing indicated that the majority of plasmids are maintained in an episomal status. Thus, plasmid-mediated transgenesis is a robust method to express foreign DNA in a promoter-specific manner in mammalian embryos and can be employed to analyze reprogramming events. The excellent technical support by E. Lemme and K. Korsawe is acknowledged. Funded by DFG.
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Choi, I., and K. H. S. Campbell. "24 EFFECTS OF HISTONE METHYLATION RELATED GENES ON EPIGENETIC REPROGRAMMING AND ZYGOTIC GENE ACTIVATION IN OVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS." Reproduction, Fertility and Development 21, no. 1 (2009): 112. http://dx.doi.org/10.1071/rdv21n1ab24.
Full textAbstract:
After fertilization, early embryo development is dependent upon maternally inherited proteins and protein synthesised from maternal mRNA until zygotic gene activation (ZGA) occurs. The transition of transcriptional activity from maternal to embryonic control occurs with the activation of rRNA genes and the formation of the nucleolus at the 8- to 16-cell stage that coincides with a prolonged fourth cell cycle in bovine and ovine embryos. However, previous studies have reported a shift in the longest cell cycle (fifth cell cycle) in bovine somatic cell nuclear transfer (SCNT) embryos, suggesting that the major genome activation is delayed, possibly due to incomplete changes in chromatin structure such as hypermethylation and hypoacetylation of histone (Memili and First 2000 Zygote 8, 87–96; Holm et al. 2003 Cloning Stem Cells 5, 133–142). Although global gene expression profile studies have been carried out in somatic cell nuclear transfer embryos, little is known about the expression of genes which can alter chromatin structure in early embryo development and possibly effect ZGA. To determine whether epigenetic reprogramming of donor nuclei affected ZGA and expression profiles in SCNT embryos, ZBTB33 (zinc finger and BTB domain containing 33, also known as kaiso, a methy-CpG specific repressor), BRG1(brahma-related gene 1, SWI/SNF family of the ATP-dependent chromatin remodeling complexes), JMJD1A (jumonji domain containing 1A, H3K9me2/1-specific demethylase), JMJD1C (putative H3K9-specific demethylase), and JMJD2C (H3K9me3-specific demethylase) were examined by RT-PCR at different developmental stages [germinal vesicle (GV), metaphase II (MII), 8- to 16-cell, 16- to 32-cell, and blastocyst in both parthenogenetic and SCNT embryos]. All genes were detected in parthenogenetic and SCNT blastocyts, and ZBTB33 was also expressed in all embryos at all stages tested. However, the onset of expression of JMJD1C, containing POU5F1 binding site at 5′-promoter region and BRG1 required for ZGA are delayed in SCNT embryos as compared to parthenotes (16- v. 8-cell, and blastoocyst v. 16-cell stage). Furthermore, JMJD2C containing NANOG binding sites at the 3′-flanking region was expressed in GV and MII oocytes and parthenogenetic blastocysts, whereas in SCNT embryos, JMJD2C was only observed from the 16-cell stage onwards. Interestingly, JMJD1A, which is positively regulated by POU5F1, was not detected in GV and MII oocytes but was present in blastocyst stage embryos of both groups. Taken together, these results suggest that incomplete epigenetic modifications of genomic DNA and histones lead to a delayed onset of ZGA which may affect further development and establishment of totipotency. Subsequently, aberrant expression patterns reported previously in SCNT embryos may be attributed to improper expression of histone H3K9 and H3K4 demethylase genes during early embryo development.
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Svoboda, Petr. "Mammalian zygotic genome activation." Seminars in Cell & Developmental Biology 84 (December 2018): 118–26. http://dx.doi.org/10.1016/j.semcdb.2017.12.006.
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Li, J., Y. Du, P. M. Kragh, S. Purup, K. Villemoes, A. M. Pedersen, A. L. Jørgensen, L. Bolund, H. M. Yang, and G. Vajta. "42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A." Reproduction, Fertility and Development 20, no. 1 (2008): 101. http://dx.doi.org/10.1071/rdv20n1ab42.
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Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells. Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells
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Berg, D. K., S. E. Beaumont, and P. L. Pfeffer. "168 miRNA LEVELS DURING BOVINE PREIMPLANTATION EMBRYONIC DEVELOPMENT." Reproduction, Fertility and Development 20, no. 1 (2008): 164. http://dx.doi.org/10.1071/rdv20n1ab168.
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MicroRNAs (miRNAs) are a class of naturally occurring non-coding RNAs that play a role in gene regulation. They are highly conserved, single-stranded RNAs, 22 nucleotides in length, that are cleaved from larger inactive hairpin precursor transcripts, and use the RNA interference-related pathways to repress their mRNA targets. They play diverse regulatory roles in cellular proliferation, morphogenesis, apoptosis, and differentiation. Maternal miRNAs are crucial for early mammalian development (Murchison et al. 2007 Genes Dev. 21, 682–693; Tang et al. 2007 Genes Dev. 21, 655–648), while sperm-borne miRNAs do not contribute significantly to miRNAs in the zygote (Amanai et al. 2006 Biol. Reprod. 75, 877–884). Our objective was to identify miRNAs that are expressed during bovine in vitro oocyte maturation (MII) and blastocyst stages as well as during parthenogenic development. MII oocytes (n = 1680) were generated from abattoir-derived oocytes and matured in vitro for 24 h. Cumulus cells were removed and the first polar body was visually assessed before the oocytes were frozen in liquid N2. Parthenogenic blastocysts (n = 575) were produced using ionomycin/6DMAP activation, and IVF blastocysts (n = 1150) were produced using standard in vitro fertilization followed by in vitro culture in synthetic oviduct fluid (Thompson et al. 2000 J. Reprod. Fertil. 118, 47–55). Blastocysts (grades 1 and 2) were selected on Day 7 post-activation/insemination and frozen in liquid N2. RNA was isolated using the mirVana miRNA isolation kit (Ambion, Scoresby, Victoria, Australia). miRNAs were quantified using the TaqMan� MicroRNA Human Panel-Early Access Kit (Applied Biosystems, Scoresby, Victoria, Australia) following the manufacturer's protocol. Absolute copy numbers per embryo were estimated. Of the 157 miRNAs in the panel, 102, 136, and 118 were detected above background in oocytes, IVF, and parthenogenic blastocysts, respectively. Only 28 miRNAs were present at over 100 copies in MII oocytes, with maximum levels reaching 1300 copies. Levels were generally much higher at blastocyst stages, with 21 miRNAs present at more than 10 000 copies. miR-16 was one of the most abundant miRNAs in all samples tested. Copy numbers per blastomere cell were 5-fold higher in IVF blastocysts compared to parthegenotic blastocysts for miR-19a, 21, and 30b. The low copy numbers of mature miRNAs before embryonic genome activation may have implications for somatic cell nuclear transfer experiments in that exogenously added miRNAs from the donor cell could impact on the embryonic gene expression profiles.
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Spate, L. D., B. K. Redel, and R. S. Prather. "82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT." Reproduction, Fertility and Development 28, no. 2 (2016): 170. http://dx.doi.org/10.1071/rdv28n2ab82.
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Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).
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Jukam, David, S. Ali M. Shariati, and Jan M. Skotheim. "Zygotic Genome Activation in Vertebrates." Developmental Cell 42, no. 4 (August 2017): 316–32. http://dx.doi.org/10.1016/j.devcel.2017.07.026.
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Schulz, Katharine N., and Melissa M. Harrison. "Mechanisms regulating zygotic genome activation." Nature Reviews Genetics 20, no. 4 (December 20, 2018): 221–34. http://dx.doi.org/10.1038/s41576-018-0087-x.
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Popken, J., A. Graf, S. Krebs, H. Blum, T. Guengoer, V. Zakhartchenko, E. Wolf, and T. Cremer. "82 STRUCTURAL REMODELLING OF THE NUCLEAR ENVELOPE IN BOVINE PRE-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 27, no. 1 (2015): 134. http://dx.doi.org/10.1071/rdv27n1ab82.
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In the present study, we investigated the changes of the nuclear envelope and its underlying lamina, as well as features of higher order chromatin organisation in bovine embryos generated by in vitro fertilization during pre-implantation development. We used super-resolution, 3-dimensional structured illumination microscopy combined with 2-colour immunostaining of the nucleoporin Nup153 and lamin B serving as markers for nuclear pore complexes (NPC) and the nuclear lamina, respectively. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). We examined 20 nuclei for the zygote (10 male pronuclei and 10 female pronuclei; n = 10) and the blastocyst (10 trophectoderm and 10 inner cell mass nuclei; n = 1) stage, and 10 nuclei for each the 2-cell (n = 5), 4-cell (n = 3), 8-cell (n = 2), 19-cell (n = 1), and morula (n = 1) stages. We report 4 major findings: (1) At the onset of major genome activation (MGA) nuclei showed a peripheral location of chromosome territories (CT), separated by wide IC channels and surrounding a major lacuna depleted of chromatin. The NPC were exclusively present at sites where DAPI-stained DNA contacted the nuclear lamina, whereas extended lamina regions without such contacts lacked NPC. In post-MGA nuclei, the CT formed a higher order chromatin network distributed throughout the entire nuclear space and the major lacuna disappeared. In line with a switch to a ubiquitous lining of DNA at the lamina, NPC were also uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions that control the integration of NPC into the nuclear envelope. (2) The switch from maternal to embryonic production of mRNA was accompanied by an increased amount of nuclear lamina invaginations covered with NPC, which may serve the increased demands of mRNA export and protein import. (3) Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Because an increase in these lamin B positive structures occurred in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size throughout bovine pre-implantation development. (4) Throughout the cytoplasm, randomly distributed extranuclear clusters of Nup153 without associated lamin B were frequently observed from the zygote stage up to MGA. These clusters may represent a deposit of maternal Nup153 and likely other nucleoporines not studied here. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced RNA before MGA, which increased strongly at the initiation of embryonic NUP153 expression at MGA. After MGA, these clusters were exclusively located at or near the nuclear border and were no longer present at the morula stage and later. In conclusion, our findings demonstrate the dynamic adaptation of the nuclear envelope to the special needs of bovine pre-implantation development and show the necessity of chromatin association for the integration of nuclear pores into the nuclear envelope.
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Anderson, Sarah N., Cameron S. Johnson, Joshua Chesnut, Daniel S. Jones, Imtiyaz Khanday, Margaret Woodhouse, Chenxin Li, Liza J. Conrad, Scott D. Russell, and Venkatesan Sundaresan. "The Zygotic Transition Is Initiated in Unicellular Plant Zygotes with Asymmetric Activation of Parental Genomes." Developmental Cell 43, no. 3 (November 2017): 349–58. http://dx.doi.org/10.1016/j.devcel.2017.10.005.
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Peng, Hui, Xiujiao Lin, Wenhao Li, and Wenchang Zhang. "Expression and localization of Nlrp4g in mouse preimplantation embryo." Zygote 23, no. 6 (November 11, 2014): 846–51. http://dx.doi.org/10.1017/s0967199414000525.
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SummaryThe Nlrp gene family contains 20 members and plays a pivotal role in the innate immune and reproductive systems in the mouse. During evolution, seven Nlrp4 gene copies (named from Nlrp4a to Nlrp4g). Nlrp4a–Nlrp4g have arisen that display specific or preferential ovarian expression patterns. However, the expression pattern and localization of Nlrp4g in mouse preimplantation embryo development are unknown. Here we report that Nlrp4g was highly expressed in mature oocytes and zygotes, then downregulated and not detected after the 2-cell embryo stage. NLRP4G protein remained present through the blastocyst stage and was mainly localized in the cytoplasm. Furthermore, overexpression of Nlrp4g in zygotes did not affect normal development in terms of the rate of blastocyst formation. These results provide the first evidence that NLRP4G is a maternal factor that may play essential role during zygotic genome activation in the mouse.
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Pálfy, Máté, Shai R. Joseph, and Nadine L. Vastenhouw. "The timing of zygotic genome activation." Current Opinion in Genetics & Development 43 (April 2017): 53–60. http://dx.doi.org/10.1016/j.gde.2016.12.001.
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Lee, Miler T., Ashley R. Bonneau, and Antonio J. Giraldez. "Zygotic Genome Activation During the Maternal-to-Zygotic Transition." Annual Review of Cell and Developmental Biology 30, no. 1 (October 11, 2014): 581–613. http://dx.doi.org/10.1146/annurev-cellbio-100913-013027.
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Memili, Erdogan, and Neal L. First. "Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species." Zygote 8, no. 1 (February 2000): 87–96. http://dx.doi.org/10.1017/s0967199400000861.
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Early embryonic development is largely dependent on maternal RNAs and proteins synthesised during oogenesis. Zygotic transcription is an essential event that occurs at a species-specific time after fertilisation. In the absence of zygotic transcription the embryo dies since it can no longer support requirements for successful embryo development. Molecular genetics of gene expression during early embryogenesis, especially in the bovine species, remain one of the unsolved questions in modern biology. Earlier studies suggested that embryonic transcription in cattle begins at the late 4-cell or 8-cell stage. However, more recent studies suggest that bovine zygotes and 2-cell embryos are both transcriptionally and translationally active. Moreover, changes in chromatin structure due to acetylation of core histones and DNA replication play important roles in the regulation of zygotic/embryonic gene expression. This review will summarise results of recent studies about the timing and mechanisms of zygotic/embryonic gene expression in cattle. In addition, terminology in the literature regarding gene expression during early embryogenesis will be clarified. These terminologies include: ‘zygotic/embryonic gene expression’, ‘maternal to embryonic transition in control of development (MET)’ and ‘zygotic/embryonic genome activation (ZEGA)’.
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Vallot, Antoine, and Kikuë Tachibana. "The emergence of genome architecture and zygotic genome activation." Current Opinion in Cell Biology 64 (June 2020): 50–57. http://dx.doi.org/10.1016/j.ceb.2020.02.002.
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Poirier, M., D. Miskel, F. Rings, K. Schellander, and M. Hoelker. "80 Biallelic CRISPR-Cas9 editing of gene associated with coat colour in microinjected bovine zygotes reaching the blastocyst stage." Reproduction, Fertility and Development 31, no. 1 (2019): 165. http://dx.doi.org/10.1071/rdv31n1ab80.
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Successful genome editing of blastocysts using zygote microinjection with transcription activator-like effector nucleases has already been accomplished in cattle as well as a limited number of CRISPR-Cas9 microinjections of zygotes, mostly using RNA. Recent editing of the Pou5f1 gene in bovine blastocysts using CRISPR-Cas9, clarifying its role in embryo development, supports the viability of this technology to produce genome edited cattle founders. To further this aim, we hypothesise that editing of the coatomer subunit α (COPA) gene, a protein carrier associated with the dominant red coat colour phenotype in Holstein cattle, is feasible through zygote microinjection. Here, we report successful gene editing of COPA in cattle zygotes reaching the blastocyst stage, a necessary step in creating genome edited founder animals. A single guide RNA was designed to target the sixth exon of COPA. Presumptive zygotes derived from slaughterhouse oocytes by in vitro maturation and fertilization were microinjected either with the PX458 plasmid (Addgene #48138; n=585, 25ng µL−1) or with a ribonucleoprotein effector complex (n=705, 20, 50, 100, and 200ng µL−1) targeting the sixth exon of COPA. Plasmid injected zygotes were selected for green fluorescent protein (GFP) fluorescence at Day 8, whereas protein injected zygotes were selected within 24h post-injection based on ATTO-550 fluorescence. To assess gene editing rates, single Day 8 blastocysts were PCR amplified and screened using the T7 endonuclease assay. Positive structures were Sanger sequenced using bacterial cloning. For plasmid injected groups, the Day 8 blastocyst rate averaged 30.3% (control 18.1%). The fluorescence rate at Day 8 was 6.3%, with a GFP positive blastocyst rate of 1.6%, totaling 7 blastocysts. The T7 assay revealed editing in GFP negative blastocysts and morulae as well, indicating that GFP is not a precise selection tool for successful editing. In protein injection groups, the highest concentration yielded the lowest survival rates (200ng µL−1, 50.0%, n=126), whereas the lowest concentration had the highest survival rate (20ng µL−1, 79.5%, n=314). The Day 8 blastocyst rate reached an average of 25% across groups. However, no edited blastocysts were observed in the higher concentration groups (100,200ng µL−1). The highest number of edited embryos was found in the lowest concentration injected (20ng µL−1, 4/56). Edited embryos showed multiple editing events neighbouring the guide RNA target site ranging from a 12-bp insertion to a 9-bp deletion, as well as unedited sequences. Additionally, one embryo showed a biallelic 15-bp deletion of COPA (10 clones). One possible reason for the presence of only mosaic editing and this in-frame deletion could be that a working copy of COPA is needed for proper blastocyst formation and that a knockout could be lethal. Additional validation and optimization is needed to elucidate the functional role of COPA during early development and its modulation when creating founder animals.
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Arnold, G. J., K. Gegenfurtner, T. Frohlich, D. R. Deutsch, P. Salvetti, N. Forde, P. Lonergan, U. Besenfelder, and E. Wolf. "59 Selected Reaction Monitoring-Based Absolute Quantification of Developmentally Relevant Proteins in Early Bovine Embryos Reveals Differences Between In Vitro and In Vivo Embryo Culture and Between Different Maternal Metabolic Stages." Reproduction, Fertility and Development 30, no. 1 (2018): 168. http://dx.doi.org/10.1071/rdv30n1ab59.
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Early embryogenesis is a highly complex developmental process, accompanied by a plethora of changes at the morphological and molecular level. Particularly at the level of proteins, these changes are still poorly characterised and understood. During the first cleavages, the embryo depends mainly on maternal transcripts and proteins that were accumulated and stored during oogenesis until embryonic genome activation (EGA) occurs. In the bovine system, the major EGA takes place at the 8- to 16-cell stage. However, we recently demonstrated by liquid chormatography-tandem mass spectrometry (LC-MS/MS)-based holistic proteome approaches that despite transcriptional and translational silencing, the proteome of the early embryo is highly dynamic (Deutsch et al. 2014; Demant et al. 2015). Based on these findings, we established a targeted LC-MS/MS approach based on multiplexed selected reaction monitoring (mSRM), which facilitates an absolute quantification of 27 proteins relevant in early embryogenesis. Each protein is targeted by 2 independent peptides to facilitate highly reliable quantifications. Nine characteristic developmental stages from germinal vesicle oocyte to hatched blastocyst were analysed (n = 6 per stage), and absolute protein contents are reported as femtomole per embryo, with limits of quantification (LOQ) down to 100 attomoles per embryo. Based on their abundance profiles during maturation, zygote formation, and embryonic development, the 27 proteins could be grouped into 6 SOTA clusters. By principal component analysis (PCA), absolute SRM quantifications of only 9 selected proteins were shown to discriminate between all 9 developmental stages analysed, thus providing molecular fingerprints significant for each developmental stage. We used the 27-plex SRM assay as a powerful readout tool and demonstrated substantial quantitative differences between embryos derived from a well-established in vitro culture system and embryos transferred into the oviduct of living animals for 2 days (in vivo culture). Furthermore, in vivo development of embryos in animals differing in their metabolic stress levels led to significant alterations in the 27-plex SRM profiles. This work was supported by a grant to GJA from Deutsche Forschungsgemeinschaft DFG FOR1041 ‘Germ Cell Potential’ AR 362/7-1 and European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 312097 - FECUND.
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Siriaco, Giorgia, and John W. Tamkun. "A Histone Timer for Zygotic Genome Activation." Developmental Cell 26, no. 6 (September 2013): 558–59. http://dx.doi.org/10.1016/j.devcel.2013.09.015.
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Østrup, Olga, Ingrid S. Andersen, and Philippe Collas. "Chromatin-linked determinants of zygotic genome activation." Cellular and Molecular Life Sciences 70, no. 8 (September 11, 2012): 1425–37. http://dx.doi.org/10.1007/s00018-012-1143-x.
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Meier, Michael, Jenny Grant, Amy Dowdle, Amarni Thomas, Jennifer Gerton, Philippe Collas, Justin M. O'Sullivan, and Julia A. Horsfield. "Cohesin facilitates zygotic genome activation in zebrafish." Development 145, no. 1 (November 20, 2017): dev156521. http://dx.doi.org/10.1242/dev.156521.
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Gentsch, George E., Nick D. L. Owens, and James C. Smith. "The Spatiotemporal Control of Zygotic Genome Activation." iScience 16 (June 2019): 485–98. http://dx.doi.org/10.1016/j.isci.2019.06.013.
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