bims-mazytr Biomed News
on Maternal‐to‐zygotic transition
Issue of 2025–06–01
eleven papers selected by
川一刀
  1. CUT&Tag for Transcription Factor Binding Profiles During Mammalian Zygotic Genome Activation.
  2. Chromatin Analyses in Mouse Oocytes and Preimplantation Embryos.
  3. Quantifying Nascent Transcription in Early Embryogenesis.
  4. Hi-C Protocol for Studying Chromatin Spatial Organization in Early Zebrafish Development.
  5. HiChIP to Study 3D Genome Organization in Xenopus tropicalis Embryos.
  6. Functional Characterization and Heterogeneity Analysis of Ribosomal Proteins in Mouse Preimplantation Embryos.
  7. Monitoring miRNA Expression and Activity in Mammalian Oocytes and Early Embryos.
  8. Imaging Translation in Early Embryo Development.
  9. A sperm-oocyte protein partnership required for egg activation in Caenorhabditis elegans.
  10. Disrupted MOS signaling alters meiotic cell cycle regulation and the egg transcriptome.
  11. Role of ZBED3 in PALD1/PIP2- dependent calcium homeostasis during oocyte maturation.
  1. Methods Mol Biol. 2025 ;2923 77-87
    CUT&Tag for Transcription Factor Binding Profiles During Mammalian Zygotic Genome Activation.
    Wataru Kobayashi, Kikuë Tachibana.
      Zygotic genome activation (ZGA) triggered by transcription factors (TFs) is a critical event to facilitate subsequent embryonic development. However, the paucity of material poses a challenge for mapping TF binding profiles in mammalian embryos. Here, we describe a low-input Cleavage Under Targets & Tagmentation (CUT&Tag) protocol optimized for TF binding using ~300 mouse two-cell embryos to investigate ZGA. This version of CUT&Tag is an immunostaining-based protocol with manual cell transfer by pipette to avoid loss of samples and gentle washing of fragile preimplantation embryos protected by the zona pellucida.
    Keywords:  CUT&Tag; Low-input sample; Mammalian preimplantation embryo; Transcription factor; Zygotic genome activation
    DOI:  https://doi.org/10.1007/978-1-0716-4522-2_6
  2. Methods Mol Biol. 2025 ;2923 33-44
    Chromatin Analyses in Mouse Oocytes and Preimplantation Embryos.
    Zhiyuan Chen, Chunxia Zhang, Yi Zhang.
      Extensive chromatin reprogramming after fertilization is essential for successful zygotic genome activation (ZGA) and embryonic development. Traditional chromatin profiling techniques chromatin immunoprecipitation assay with sequencing (ChIP-seq) and deoxyribonuclease I hypersensitivity sequencing (DNase-seq) require large number of cells, which are not suitable for rare biological materials such as mammalian preimplantation embryos. Recent advancement of low-input epigenome profiling techniques has allowed the exploration of chromatin dynamics and functions during ZGA and early embryonic development. In this chapter, we describe two low-input methods, namely, CUT&RUN and ATAC-seq, that are efficient and robust for chromatin analyses using as few as 50-100 cells. These methods are useful for profiling histone modifications, histone variants, and chromatin accessibility in mammalian preimplantation studies.
    Keywords:  ATAC-seq; CUT&RUN; Chromatin; Low input; Oocytes; Preimplantation embryos
    DOI:  https://doi.org/10.1007/978-1-0716-4522-2_3
  3. Methods Mol Biol. 2025 ;2923 143-162
    Quantifying Nascent Transcription in Early Embryogenesis.
    Hui Chen.
      The early embryonic genome exists in a dormant state following fertilization, and it then subsequently undergoes broad activation of zygotic transcription at the early stages of development. A major challenge is the detection of newly made zygotic transcripts and the determination of their activation onset time due to the presence of large and predominantly maternal pool of RNAs. Here we describe a detailed method to measure the zygotic transcription during zygotic genome activation (ZGA) of Xenopus early embryos using metabolic labeling of nascent transcripts with 5-ethynyl uridine (5-EU) followed by purifying and sequencing the nascent EU-RNAs (EU-RNA-seq). This method is highly sensitive in detecting early zygotic transcripts that are not detected by total RNA-seq and determines the actual onset time of transcriptional activation for zygotic genes. The method is applicable to a wide variety of embryonic model systems and has already afforded novel insights into gene regulation in early embryogenesis.
    Keywords:  5-ethynyl uridine (5-EU); Early embryogenesis; Nascent transcription; RNA sequencing (RNA-seq); Transcriptome; Zygotic genome activation
    DOI:  https://doi.org/10.1007/978-1-0716-4522-2_9
  4. Methods Mol Biol. 2025 ;2923 45-61
    Hi-C Protocol for Studying Chromatin Spatial Organization in Early Zebrafish Development.
    Sergey V Ulianov, Mariia Bazarevich, Natalia V Petrova, Daria Onichtchouk.
      Hi-C is a high-throughput technique which allowed to discover three-dimensional chromatin structures in interphase nuclei. The questions of how these structures emerge, and how genome 3D organization can influence its function, are still poorly understood. Research in early embryos is uniquely suited to shed the light to these questions: In animal embryos, three-dimensional structures in chromatin are born during the major zygotic genome activation (ZGA) wave or shortly after it. Our own study addresses causal connection between events coinciding with ZGA and chromatin folding, using zebrafish early embryos as a model system. Here, we provide a detailed description of our Hi-C protocol adapted for zebrafish embryos.
    Keywords:  Danio rerio; Genome spatial organization; Hi-C; Zebrafish; Zygotic genome activation
    DOI:  https://doi.org/10.1007/978-1-0716-4522-2_4
  5. Methods Mol Biol. 2025 ;2923 63-74
    HiChIP to Study 3D Genome Organization in Xenopus tropicalis Embryos.
    Amina O Hussein, Ira L Blitz, Ken W Y Cho.
      During zygotic genome activation, the embryonic genome starts to organize into three-dimensional (3D) structures such as topologically associated domains and chromatin loops, which are essential for regulating gene expression during development. HiChIP is a powerful tool for investigating 3D genome organization by identifying proteins associated with long-range chromatin interactions. In this chapter, we provide a protocol for preparing HiChIP libraries from Xenopus tropicalis embryos.
    Keywords:  Gastrulation; Germ layer formation; HiChIP; TADs; Xenopus; Zygotic genome activation
    DOI:  https://doi.org/10.1007/978-1-0716-4522-2_5
  6. FASEB J. 2025 Jun 15. 39(11): e70662
    Functional Characterization and Heterogeneity Analysis of Ribosomal Proteins in Mouse Preimplantation Embryos.
    Seung Jae Lee, Dayeon Kim, Gwidong Han, Seung Pyo Hong, Inchul Choi, Chunghee Cho.
      Translational control is important during the mammalian preimplantation phase, when maternal RNAs and proteins are degraded and de novo synthesis of RNAs and proteins increases. Proteins are synthesized in ribosomes, which are assembled from ~82 ribosomal proteins (RPs). The function of ribosomes varies depending on the resident RPs, suggesting that ribosome heterogeneity can lead to functional specialization. Only a few studies have investigated the function of RPs during preimplantation embryonic development. Here, we performed functional analyses on six RP-encoding genes-Rpl4, Rps9, Rps11, Rpl13a, Rpl19, and Rpl39-in mouse preimplantation embryos. Knockdown (KD) of each of these RP genes, except Rpl39, affected morula-to-blastocyst transition, producing phenotypes that varied somewhat in their details. Rpl4-, Rpl13a-, and Rpl19-KD embryos showed fragmentation and strong arrest of cell proliferation, whereas Rps9- and Rps11-KD embryos showed severe fragmentation with relatively weak arrest of cell proliferation. In the case of Rpl39, single-KD embryos developed normally, but double-KD embryos with its paralog Rpl39-like (Rpl39l) inhibited normal blastocyst development. Protein misfolding signals were also activated in Rpl39-KD and Rpl39l + Rpl39 double-KD embryos, confirming a previous finding that RPL39 and RPL39L are associated with ribosome exit tunnels. Our results suggest the presence of different groups of proteins that require an RPL39-containing ribosome or RPL39/RPL39L-containing ribosome for correct folding in early embryos. Taken together, the results of the present study demonstrate that ribosomal proteins are fundamentally important for normal blastocyst formation and development, but not all ribosomal proteins contribute equally to embryonic development, providing a novel example of ribosome heterogeneity in preimplantation embryos.
    Keywords:  de novo protein synthesis; morula to blastocyst transition; mouse preimplantation embryo; protein folding; ribosomal protein; ribosome heterogeneity
    DOI:  https://doi.org/10.1096/fj.202500574RR
  7. Methods Mol Biol. 2025 ;2923 181-194
    Monitoring miRNA Expression and Activity in Mammalian Oocytes and Early Embryos.
    Shubhangini Kataruka, Petr Svoboda.
      MicroRNAs (miRNAs), genome-encoded small RNAs associated with Argonaute proteins, are important negative regulators of gene expression in mammalian cells. miRNAs usually partially base pair with mRNAs, suppress their translation, and destabilize them. Sufficient miRNA abundance is an important factor for efficient target repression. Experimental evidence suggests that oocyte growth causes a dilution effect, which reduces concentrations of maternal miRNAs and renders them functionally inefficient. Consequently, efficient target repression is retained only by those maternal miRNAs which achieve a favorable miRNA:mRNA stoichiometry. Here, we provide protocols for PCR-based quantification of miRNAs and luciferase reporter-based analysis of their activity in mammalian oocytes and early embryos.
    Keywords:  Microinjection; NanoLuc; Oocyte; Zygote; mRNA reporter; miRNAs
    DOI:  https://doi.org/10.1007/978-1-0716-4522-2_11
  8. Methods Mol Biol. 2025 ;2923 215-229
    Imaging Translation in Early Embryo Development.
    Pierre Bensidoun, Morgane Verbrugghe, Mounia Lagha.
      The ultimate output of gene expression is to ensure that proteins are synthesized at the right levels, locations, and timings. Recently different imaging-based methods have been developed to visualize the translation of single mRNA molecules. These methods rely on signal amplification with the introduction of an array of a short peptide sequence (a tag such as SunTag), recognized by a genetically encodable single-chain antibody (a detector such as scFv). In this chapter, we discuss such methods to image and quantify translation dynamics in the early Drosophila embryo and provide examples based on a twist-32XSunTag reporter. We outline a step-by-step protocol to light-up translation in living embryos. We also detail a combinatorial strategy in fixed samples (smFISH-IF), allowing to distinguish single mRNA molecules engaged in translation.
    Keywords:  Cell biology; Developmental biology; Drosophila; Embryo; Gene expression; Live imaging; SunTag/scFv; Translation; smFISH
    DOI:  https://doi.org/10.1007/978-1-0716-4522-2_13
  9. Development. 2025 May 30. pii: dev.204674. [Epub ahead of print]
    A sperm-oocyte protein partnership required for egg activation in Caenorhabditis elegans.
    Tatsuya Tsukamoto, Ji Kent Kwah, Mark E Zweifel, Naomi Courtemanche, Micah D Gearhart, Katherine M Walstrom, Aimee Jaramillo-Lambert, David Greenstein.
      Fertilization triggers the completion of female meiosis and launches the oocyte-to-embryo transition. C. elegans spe-11 is one of the few known paternal-effect embryonic lethal genes. We report that the sperm protein, SPE-11, forms a complex with an oocyte protein, OOPS-1 (Oocyte Partner of SPE-11) at fertilization, and that the protein complex is required for the completion of meiosis, the block to polyspermy, and eggshell formation. Consistent with the molecular interaction of their encoded proteins, oops-1 and spe-11 exhibit indistinguishable null phenotypes in which fertilized oocytes arrest in meiosis I or meiosis II or fail to complete the actin-based process of meiotic cytokinesis. Biochemical analysis shows that the complex binds F-actin in the absence of other proteins and inhibits the nucleation of actin filaments at substoichiometric concentrations. Both OOPS-1 and SPE-11 are intrinsically disordered proteins that are highly phosphorylated, and biochemical and genetic experiments define interactions with sperm-specific protein phosphatase 1 homologs GSP-3/4. Genetic results suggest that the cortical EGG complex recruits the OOPS-1-SPE-11 complex at fertilization, which promotes meiotic cytokinesis and in turn activates synthesis of the eggshell.
    Keywords:  And actin regulation; Egg activation; Fertilization; Meiotic cytokinesis; Oocyte meiosis
    DOI:  https://doi.org/10.1242/dev.204674
  10. Reproduction. 2025 May 01. pii: REP-25-0156. [Epub ahead of print]
    Disrupted MOS signaling alters meiotic cell cycle regulation and the egg transcriptome.
    Gisela Cairo, Olha Kholod, Olivia Palmer, Sophia Meytin, Brittany A Goods, Soni Lacefield.
      Mammalian female meiosis is tightly regulated to produce a developmentally competent egg. Oocytes enter meiosis in the fetal ovary and then arrest at prophase I until sexual maturation. Upon hormonal stimulation, a subset of oocytes resumes meiosis. Oocytes then complete meiosis I, enter metaphase II, and arrest until fertilization, a process essential for egg competency. The MOS kinase is a key regulator of the metaphase II arrest, activating the MAPK signaling cascade. Loss of MOS in female mice disrupts the maintenance of the metaphase II arrest, with some eggs extruding two polar bodies and some dividing beyond anaphase II. To investigate the consequences of the Mos deletion, we performed live imaging and found that mos-/- eggs exhibit transient chromosome separation events in meiosis I, suggesting a role for MOS in coordinating the timing of meiotic divisions. Further analysis showed that new transcription is required for mos-/- eggs to undergo additional divisions but not for second polar body extrusion. Surprisingly, single-egg sequencing revealed extensive differences in gene expression between wildtype and mos-/- eggs, including those with only one polar body. Many differentially expressed genes were involved in cell cycle regulation, including Aurka, Bub3, and Cdk7. Upregulated pathways included metabolism of RNA, transcription, and neddylation. Furthermore, the gene expression profile of mos-/- eggs was markedly different from that of chemically activated wildtype eggs. Our findings demonstrate that MOS plays a crucial role in meiotic cell cycle regulation and helps ensure that the egg maintains the proper transcriptome necessary for developmental competence.
    DOI:  https://doi.org/10.1530/REP-25-0156
  11. Cell Biosci. 2025 May 24. 15(1): 68
    Role of ZBED3 in PALD1/PIP2- dependent calcium homeostasis during oocyte maturation.
    Danyu Ni, Qijun Xie, Yuting Chen, Yi Wei, Peng Lang, Xiaodan Shi, Ye Yang, Xiufeng Ling, Chun Zhao.
      Zinc Finger BED-Type Containing 3 (ZBED3) had been shown to be a novel component of the subcortical maternal complex (SCMC). In previous reports, ZBED3 depletion leads to asymmetric zygotic division and aberrant distribution of organelles in both oocytes and zygotes. However, the precise mechanism through which ZBED3 exerts its effects remains to be elucidated. To fill this gap, in this study, we generated Zbed3 gene knockout mice by using CRISPR/cas9 gene-editing technique to generate homozygous Zbed3-/- female mice. A series of previously unreported phenotypes in oocytes were observed, including decreased fertility, abnormal spindle formation and migration, increased polyspermic fertilization, abnormal distribution of cortical granules (CGs), and disrupted calcium oscillations. To investigate the molecular mechanisms underlying the function of ZBED3 during oocyte maturation, we employed miniTurbo biotin ligase-based proximity labeling combined with mass spectrometry to identify protein interactomes in transfected HEK293 cells. OF the 187 ZBED3-interacting proteins, paladin 1 containing a phosphatase domain (PALD1) and E3 ubiquitin ligase makorin-1 (MKRN1) exhibited the highest fold changes and were subsequently validated. ZEBD3 suppressed PALD1 levels by enhancing its degradation via the ubiquitination-proteasome pathway. Depletion of Zbed3 results in an abnormal accumulation of PALD1. The ectopic overexpression of PALD1 recapitulates the phenotypic defects observed in Zbed3-deficient oocytes and early embryos. Moreover, knockdown of PALD1 partially rescued the oocyte maturation defects induced by Zbed3 depletion. Paladin is an endosomal phosphatidylinositol 4,5-bisphosphate (PIP2) phosphatase which directly modulates phosphoinositide metabolism by catalyzing the removal of phosphate groups from phosphoinositides. Furthermore, PALD1 overexpression reduced Ca2+ release from the endoplasmic reticulum (ER) by inhibiting its downstream target PIP2. Our study demonstrates that ZBED3 may regulate PIP2 protein levels by modulating the ubiquitin-proteasomal degradation of PALD1, thereby influencing oocyte maturation and providing a novel approach for assessing oocyte quality and developmental potential.
    Keywords:  CRISPR/cas9; Calcium homeostasis; F-actin assembly; Oocyte maturation; PALD1; PIP2; Subcortical maternal complex (SCMC); ZBED3
    DOI:  https://doi.org/10.1186/s13578-025-01404-y
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