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Genesis (New York, N.Y. : 2000) Oct 2005Following fertilization, a number of molecular events are triggered in the mammalian zygote. As biochemical studies using mammalian gametes and zygotes have inherent...
Following fertilization, a number of molecular events are triggered in the mammalian zygote. As biochemical studies using mammalian gametes and zygotes have inherent difficulties, the molecular nature of these processes is currently unclear. We have developed a method to visualize these events. In vitro transcribed mRNAs encoding for proteins fused with green fluorescent protein were microinjected into oocytes or embryos and fluorescence signals were observed. Using this technique we succeeded in obtaining images of the DNA methylation status in living mouse and rabbit embryos. Moreover, time-lapse images were acquired of spindle and nuclear formation during second meiosis and first mitosis. Importantly, the microinjected embryos developed to the normal offspring even after observation, suggesting that the technique is relatively noninvasive. Thus, our method may help elucidate the molecular aspects of fertilization and preimplantation development and, based on the real-time genetic and epigenetic status, could become a tool to select "good quality" embryos before implantation.
Topics: Animals; DNA Methylation; Embryonic Development; Green Fluorescent Proteins; Luminescent Agents; Mice; Microscopy, Fluorescence; Protein Biosynthesis; RNA, Messenger; Rabbits; Recombinant Fusion Proteins; Transfection; Zygote
PubMed: 16100711
DOI: 10.1002/gene.20158 -
International Journal of Molecular... Aug 2022Maternal-to-zygotic transition (MZT) of the control of early post-fertilization development is a key-event conditioning the fate of the future embryo, fetus and newborn.... (Review)
Review
Maternal-to-zygotic transition (MZT) of the control of early post-fertilization development is a key-event conditioning the fate of the future embryo, fetus and newborn. Because of the relative paucity of data concerning human embryos, due to ethical concerns and the poor availability of human embryos donated for research, most data have to be derived from animal models, among which those obtained using mouse embryos are most prevalent. However, data obtained by studies performed in non-mammalian specie can also provide useful information. For this reason, this review focuses on similarities and differences of MZT control mechanisms in humans and other species, with particular attention to the mouse. A number of molecular pathways controlling MZT in mice and humans are compared, pointing out those that could be at the origin of further focused experimental studies and the development of new diagnostic tools based on the translational medicine principles. Data concerning possible candidate molecules to be included in these studies are identified.
Topics: Animals; Embryo, Mammalian; Embryonic Development; Gene Expression Regulation, Developmental; Humans; Mice; Zygote
PubMed: 35955697
DOI: 10.3390/ijms23158562 -
DNA Repair Aug 2006In the mouse, the paternal post-meiotic chromatin is assumed to be devoid of DNA repair after nuclear elongation and protamine-induced compaction. Hence, DNA lesions... (Comparative Study)
Comparative Study
In the mouse, the paternal post-meiotic chromatin is assumed to be devoid of DNA repair after nuclear elongation and protamine-induced compaction. Hence, DNA lesions induced thereafter will have to be restored upon gamete fusion in the zygote. Misrepair of such lesions often results in chromosome type aberrations at the first cleavage division, suggesting that the repair event takes place prior to S-phase. During this stage of the zygotic cell cycle, the paternal chromatin transits from a protamine- to a nucleosome-based state. We addressed the question whether the canonical signalling pathway to DNA double strand breaks (DSBs), the phosphorylated form of histone H2AX (gammaH2AX) is active during chromatin restructuring of the male genetic complement in the zygote. Here, we describe the detailed characterization of gammaH2AX signalling in the early stages of zygotic development up to the appearance of the pronuclei. We have found the gammaH2AX signalling pathway to be already active during sperm chromatin remodelling after gamete fusion in a dose dependent manner, reflecting the amount of DSBs present in the sperm nucleus after in vivo male irradiation. Using DNA damaging compounds to induce lesions in the early zygote, differences in DSB sensitivity and gammaH2AX processing between paternal and maternal chromatin were found, suggesting differences in DNA repair capacity between the parental chromatin sets.
Topics: Animals; Bleomycin; Chromatin Assembly and Disassembly; DNA Repair; Etoposide; Histones; Male; Mice; Microscopy, Fluorescence; Sex Factors; Signal Transduction; Spermatozoa; Zygote
PubMed: 16837249
DOI: 10.1016/j.dnarep.2006.05.043 -
Methods in Molecular Biology (Clifton,... 2019The rat is one the most widely used laboratory animal species in many aspects of biomedical research, including the production of genetically engineered animal models to...
The rat is one the most widely used laboratory animal species in many aspects of biomedical research, including the production of genetically engineered animal models to study human diseases and conditions. In addition to in vitro fertilization (IVF), the ability to grow IVF-derived or in vivo-collected zygotes to a desired preimplantation stage (zygote to blastocyst) entirely in vitro has a great importance for studies of developmental biology and genetic modification of laboratory rats for biomedical research. Although embryo biotechnologies are required to study or manipulate the genome effectively, such technologies for rat preimplantation embryos are not currently as successful as they are in the mouse. Here we provide a brief history of the development of rat in vitro culture systems and a step-by-step protocol to produce rat blastocyst stage embryos from zygotes under in vitro conditions from commonly used laboratory rat strains in biomedical research.
Topics: Animals; Blastocyst; Culture Media; Embryo Culture Techniques; Embryonic Development; Female; Fertilization in Vitro; Humans; Rats; Zygote
PubMed: 31230271
DOI: 10.1007/978-1-4939-9566-0_3 -
Journal of Visualized Experiments : JoVE Sep 2017In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example,...
In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.
Topics: Arabidopsis; Cell Differentiation; Microscopy, Polarization; Ovule; Zygote
PubMed: 28930998
DOI: 10.3791/55975 -
Current Topics in Developmental Biology 2016Zygotic genome activation (ZGA, a.k.a. zygotic gene activation) is a critical event in development, when the paternally derived genome and maternally derived genome... (Review)
Review
Zygotic genome activation (ZGA, a.k.a. zygotic gene activation) is a critical event in development, when the paternally derived genome and maternally derived genome begin to be activated and transcribed after fertilization. Major ZGA occurs at the two-cell stage in mice and the four- to eight-cell stage in human preimplantation embryos. It has been thought that ZGA exists to provide RNAs and proteins supporting embryonic development after supplies stored in oocytes are used up; however, this paradigm does not seem to explain recent findings. For example, many ZGA genes-once activated-are quickly turned off, and thus ZGA forms a transient wave of transcriptional activation. In addition, ZGA genes are not evolutionarily conserved. In this review, we address these issues by focusing on Zscan4 (zinc finger and SCAN domain-containing 4), which was identified for its specific expression in preimplantation embryos during ZGA. Detailed molecular analyses of Zscan4 expression and function have revealed common features of Zscan4-associated events (Z4 events) in mouse embryonic stem cells and ZGA in preimplantation embryos. One feature is a rapid derepression and rerepression of constitutive heterochromatin, which includes pericentromeric major satellites and telomeres, and facultative heterochromatin, which includes retrotransposons and Z4 event-associated genes. We propose that the Z4 event superimposed on ZGA plays a critical role in the maintenance of genome and chromosome integrity in preimplantation embryos by promoting correction of DNA damage and chromosome abnormalities.
Topics: Animals; DNA Damage; Evolution, Molecular; Gene Expression Regulation, Developmental; Genome; Humans; Transcription Factors; Zygote
PubMed: 27475850
DOI: 10.1016/bs.ctdb.2016.04.004 -
Advances in Genetics 2016Embryo development commences with the fusion of two terminally differentiated haploid gametes into the totipotent fertilized egg, which through a series of major... (Review)
Review
Embryo development commences with the fusion of two terminally differentiated haploid gametes into the totipotent fertilized egg, which through a series of major cellular and molecular transitions generate a pluripotent cell mass. The activation of the zygotic genome occurs during the so-called maternal to zygotic transition and prepares the embryo for zygotic takeover from maternal factors, in the control of the development of cellular lineages during differentiation. Recent advances in next generation sequencing technologies have allowed the dissection of the genomic and epigenomic processes mediating this transition. These processes include reorganization of the chromatin structure to a transcriptionally permissive state, changes in composition and function of structural and regulatory DNA-binding proteins, and changeover of the transcriptome as it is overhauled from that deposited by the mother in the oocyte to a zygotically transcribed complement. Zygotic genome activation in zebrafish occurs 10 cell cycles after fertilization and provides an ideal experimental platform for elucidating the temporal sequence and dynamics of establishment of a transcriptionally active chromatin state and helps in identifying the determinants of transcription activation at polymerase II transcribed gene promoters. The relatively large number of pluripotent cells generated by the fast cell divisions before zygotic transcription provides sufficient biomass for next generation sequencing technology approaches to establish the temporal dynamics of events and suggest causative relationship between them. However, genomic and genetic technologies need to be improved further to capture the earliest events in development, where cell number is a limiting factor. These technologies need to be complemented with precise, inducible genetic interference studies using the latest genome editing tools to reveal the function of candidate determinants and to confirm the predictions made by classic embryological tools and genome-wide assays. In this review we summarize recent advances in the characterization of epigenetic regulation, transcription control, and gene promoter function during zygotic genome activation and how they fit with old models for the mechanisms of the maternal to zygotic transition. This review will focus on the zebrafish embryo but draw comparisons with other vertebrate model systems and refer to invertebrate models where informative.
Topics: Animals; Gene Expression Regulation, Developmental; Genome; Humans; Transcription, Genetic; Transcriptome; Zebrafish; Zygote
PubMed: 27503357
DOI: 10.1016/bs.adgen.2016.05.001 -
Journal of Plant Research Jul 2022Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C crops because S. viridis has several desirable traits,...
Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C crops because S. viridis has several desirable traits, such as short generation time, prolific seed production and a small genome size. These advantages are well suited for investigating molecular mechanisms in angiosperms, especially C crop species. Here, we report a procedure for isolating gametes and zygotes from S. viridis flowers. To isolate egg cells, ovaries were harvested from unpollinated mature flowers and cut transversely, which allowed direct access to the embryo sac. Thereafter, an egg cell was released from the cut end of the basal portion of the dissected ovary. To isolate sperm cells, pollen grains released from anthers were immersed in a mannitol solution, resulting in pollen-grain bursting, which released sperm cells. Additionally, S. viridis zygotes were successfully isolated from freshly pollinated flowers. Isolated zygotes cultured in a liquid medium developed into globular-like embryos and cell masses. Thus, isolated S. viridis gametes, zygotes and embryos are attainable for detailed observations and investigations of fertilization and developmental events in angiosperms.
Topics: Flowers; Pollen; Seeds; Setaria Plant; Zygote
PubMed: 35534650
DOI: 10.1007/s10265-022-01393-w -
Cell Dec 2016DNA demethylation, a process involving DNA repair, is critical for reprogramming of the paternal genome during the oocyte-to-zygote transition. A new study by...
DNA demethylation, a process involving DNA repair, is critical for reprogramming of the paternal genome during the oocyte-to-zygote transition. A new study by Ladstätter and Tachibana-Konwalski shows that a Chk1-mediated zygotic checkpoint monitors the cohesin-dependent repair of DNA lesions arising from DNA demethylation, which prevents zygotes carrying unrepaired lesions from entering mitosis.
Topics: Cell Cycle Checkpoints; DNA Methylation; DNA Repair; Genome; Humans; Zygote
PubMed: 27984718
DOI: 10.1016/j.cell.2016.11.049 -
Reproductive Biology Mar 2008Embryo quality related to its developmental potential is now one of the most important issues in modern embryology. It has been demonstrated that some in vitro produced... (Review)
Review
Embryo quality related to its developmental potential is now one of the most important issues in modern embryology. It has been demonstrated that some in vitro produced blastocysts fail to hatch and implant after transfer despite a normal morphology. Although embryos are able to adjust to sub-optimal culture conditions, significant changes in expression profiles of developmentally important genes have been noticed. Timing of the first zygotic cleavage is considered a non-invasive marker of embryo developmental potential and has been successfully used in human IVF programs for identifying embryos of superior quality. Early-cleaving zygotes are more likely to develop to the blastocyst stage than their late-cleaving counterparts. The timing of the first zygotic cleavage has been associated with several parameters that may affect developmental potential of the resulting embryos. The mechanism causing variation in the timing of the first zygotic cleavage has not been identified. It may be related to culture environment or to some intrinsic factors within the oocyte, the sperm or both. In this paper we discuss some of the important aspects related to the timing of the first zygotic cleavage and its influence on the developmental competence of resulting embryos.
Topics: Animals; Biomarkers; Cleavage Stage, Ovum; Embryo Culture Techniques; Fertilization in Vitro; Humans; Time Factors; Zygote
PubMed: 18432305
DOI: 10.1016/s1642-431x(12)60002-3