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Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance., Competing Interests: Declaration of competing interest There are no conflict of interest for any of the authors that there has been no duplicate publication or submission elsewhere of this work that all authors have read and approved the manuscript, are aware of the submission for publication and agree to be listed as co-authors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Little is known about the origin of germ cells in humans. We previously leveraged post-zygotic mutations to reconstruct zygote-rooted cell lineage ancestry trees in a phenotypically normal woman, termed NC0. Here, by sequencing the genome of her children and their father, we analyze the transmission of early pre-gastrulation lineages and corresponding mutations across human generations. We find that the germline in NC0 is polyclonal and is founded by at least two cells likely descending from the two blastomeres arising from the first zygotic cleavage. Analyzes of public data from several multi-children families and from 1934 familial quads confirm this finding in larger cohorts, revealing that known imbalances of up to 90:10 in early lineages allocation in somatic tissues are not reflected in mutation transmission to offspring, establishing a fundamental difference in lineage allocation between the soma and the germline. Analyzes of all the data consistently suggest that the germline has a balanced 50:50 lineage allocation from the first two blastomeres., (© 2024. The Author(s).)

The maternal-to-zygotic transition is crucial in embryonic development, marked by the degradation of maternally provided mRNAs and initiation of zygotic gene expression. However, the changes occurring at the protein level during this transition remain unclear. Here, we conducted protein profiling throughout zebrafish embryogenesis using quantitative mass spectrometry, integrating transcriptomics and translatomics datasets. Our data show that, unlike RNA changes, protein changes are less dynamic. Further, increases in protein levels correlate with mRNA translation, whereas declines in protein levels do not, suggesting active protein degradation processes. Interestingly, proteins from pure zygotic genes are present at fertilization, challenging existing mRNA-based gene classifications. As a proof of concept, we utilized CRISPR-Cas13d to target znf281b mRNA, a gene whose protein significantly accumulates within the first 2 h post-fertilization, demonstrating its crucial role in development. Consequently, our protein profiling, coupled with CRISPR-Cas13d, offers a complementary approach to unraveling maternal factor function during embryonic development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The subcortical maternal complex (SCMC) is essential for safeguarding female fertility in mammals. Assembled in oocytes, the SCMC maintains the cleavage of early embryos, but the underlying mechanism remains unclear. Here, we report that 14-3-3, a multifunctional protein, is a component of the SCMC. By resolving the structure of the 14-3-3-containing SCMC, we discover that phosphorylation of TLE6 contributes to the recruitment of 14-3-3. Mechanistically, during maternal-to-embryo transition, the SCMC stabilizes 14-3-3 protein and contributes to the proper control of CDC25B, thus ensuring the activation of the maturation-promoting factor and mitotic entry in mouse zygotes. Notably, the SCMC establishes a conserved molecular link with 14-3-3 and CDC25B in human oocytes/embryos. This study discloses the molecular mechanism through which the SCMC regulates the cell cycle in early embryos and elucidates the function of the SCMC in mammalian early embryogenesis., (© 2024. The Author(s).)

Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. Although the TFs specifying the blastocyst's lineages are well characterized, those playing earlier roles remain poorly defined. Using mouse models of the TF Nr5a2 , we show that Nr5a2 -/- embryos arrest at the early morula stage and exhibit altered lineage specification, frequent mitotic failure, and substantial chromosome segregation defects. Although NR5A2 plays a minor but measurable role during zygotic genome activation, it predominantly acts as a master regulator at the eight-cell stage, controlling expression of lineage-specifying TFs and genes involved in mitosis, telomere maintenance, and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability, and lineage specification to ensure correct morula development.

Precise spatiotemporal regulation of gene expression is of paramount importance for eukaryotic development. The maternal-to-zygotic transition (MZT) during early embryogenesis in Drosophila involves the gradual replacement of maternally contributed mRNAs and proteins by zygotic gene products. The zygotic genome is transcriptionally activated during the first 3 hours of development, in a process known as "zygotic genome activation" (ZGA), by the orchestrated activities of a few pioneer factors. Their decisive role during ZGA has been characterized in detail, whereas the contribution of chromatin factors to this process has been historically overlooked. In this review, we aim to summarize the current knowledge of how chromatin regulation impacts the first stages of Drosophila embryonic development. In particular, we will address the following questions: how chromatin factors affect ZGA and transcriptional silencing, and how genome architecture promotes the integration of these processes early during development. Remarkably, certain chromatin marks can be intergenerationally inherited, and their presence in the early embryo becomes critical for the regulation of gene expression at later stages. Finally, we speculate on the possible roles of these chromatin marks as carriers of epialleles during transgenerational epigenetic inheritance (TEI)., (© 2024. The Author(s).)

Oocyte maturation and preimplantation embryo development are critical to successful pregnancy outcomes and the correct establishment and maintenance of genomic imprinting. Thanks to novel technologies and omics studies in human patients and mouse models, the importance of the proteins associated with the cytoplasmic lattices (CPLs), highly abundant structures found in the cytoplasm of mammalian oocytes and preimplantation embryos, in the maternal to zygotic transition is becoming increasingly evident. This review highlights the recent discoveries on the role of these proteins in protein storage and other oocyte cytoplasmic processes, epigenetic reprogramming, and zygotic genome activation (ZGA). A better comprehension of these events may significantly improve clinical diagnosis and pave the way for targeted interventions aiming to correct or mitigate female fertility issues and genomic imprinting disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Zygotic genome activation (ZGA), the first transcription event following fertilization, kickstarts the embryonic program that takes over the control of early development from the maternal products. How ZGA occurs, especially in mammals, is poorly understood due to the limited amount of research materials. With the rapid development of single-cell and low-input technologies, remarkable progress made in the past decade has unveiled dramatic transitions of the epigenomes, transcriptomes, proteomes, and metabolomes associated with ZGA. Moreover, functional investigations are yielding insights into the key regulators of ZGA, among which two major classes of players are emerging: licensors and specifiers. Licensors would control the permission of transcription and its timing during ZGA. Accumulating evidence suggests that such licensors of ZGA include regulators of the transcription apparatus and nuclear gatekeepers. Specifiers would instruct the activation of specific genes during ZGA. These specifiers include key transcription factors present at this stage, often facilitated by epigenetic regulators. Based on data primarily from mammals but also results from other species, we discuss in this review how recent research sheds light on the molecular regulation of ZGA and its executors, including the licensors and specifiers., (© 2024. The Author(s).)

How barriers to gene flow arise and are maintained are key questions in evolutionary biology. Speciation research has mainly focused on barriers that occur either before mating or after zygote formation. In comparison, postmating prezygotic (PMPZ) isolation-a barrier that acts after gamete release but before zygote formation-is less frequently investigated but may hold a unique role in generating biodiversity. Here we discuss the distinctive features of PMPZ isolation, including the primary drivers and molecular mechanisms underpinning PMPZ isolation. We then present the first comprehensive survey of PMPZ isolation research, revealing that it is a widespread form of prezygotic isolation across eukaryotes. The survey also exposes obstacles in studying PMPZ isolation, in part attributable to the challenges involved in directly measuring PMPZ isolation and uncovering its causal mechanisms. Finally, we identify outstanding knowledge gaps and provide recommendations for improving future research on PMPZ isolation. This will allow us to better understand the nature of this often-neglected reproductive barrier and its contribution to speciation., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

Post-translational modification and fine-tuned protein turnover are of great importance in mammalian early embryo development. Apart from the classic protein degradation promoting ubiquitination, new forms of ubiquitination-like modification are yet to be fully understood. Here, we demonstrate the function and potential mechanisms of one ubiquitination-like modification, neddylation, in mouse preimplantation embryo development. Treated with specific inhibitors, zygotes showed a dramatically decreased cleavage rate and almost all failed to enter the 4-cell stage. Transcriptional profiling showed genes were differentially expressed in pathways involving cell fate determination and cell differentiation, including several down-regulated zygotic genome activation (ZGA) marker genes. A decreased level of phosphorylated RNA polymerase II was detected, indicating impaired gene transcription inside the embryo cell nucleus. Proteomic data showed that differentially expressed proteins were enriched in histone modifications. We confirmed the lowered in methyltransferase (KMT2D) expression and a decrease in histone H3K4me3. At the same time, acetyltransferase (CBP/p300) reduced, while deacetylase (HDAC6) increased, resulting in an attenuation in histone H3K27ac. Additionally, we observed the up-regulation in YAP1 and RPL13 activities, indicating potential abnormalities in the downstream response of Hippo signaling pathway. In summary, we found that inhibition of neddylation induced epigenetic changes in early embryos and led to abnormalities in related downstream signaling pathways. This study sheds light upon new forms of ubiquitination regulating mammalian embryonic development and may contribute to further investigation of female infertility pathology., Competing Interests: Declaration of competing interest The authors claim to have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The activation of the zygotic genome constitutes an essential process during early embryogenesis that determines the overall progression of embryonic development. Zygotic genome activation (ZGA) is tightly regulated, involving a delicate interplay of activators and repressors, to precisely control the timing and spatial pattern of gene expression. While regulators of ZGA vary across species, they accomplish comparable outcomes. Recent studies have shed light on the unanticipated roles of ZGA components both during and after ZGA. Moreover, different ZGA regulators seem to have acquired unique functional modalities to manifest their regulatory potential. In this review, we explore these observations to assess whether these are simply anecdotal or contribute to a broader regulatory framework that employs a versatile means to arrive at the conserved outcome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Damage to the development of porcine gametes and embryos caused by high temperatures (HT) is one of the main reasons for the decline in the economic benefits of the livestock industry. Zygotic genome activation (ZGA) marks the beginning of gene expression programs in mammalian pre-implantation embryos. In pigs, ZGA occurs at the 4-cell (4 C) stage, indicating that correct gene expression at this stage plays an important regulatory role in embryonic development. However, the effect of the HT environment on early porcine embryonic development and the RNA expression profile of ZGA remain unclear. In this study, we compared the RNA transcription patterns of porcine 4 C embryos under normal and HT conditions using RNA-seq and identified 326 differentially expressed genes (DEGs). These changes were mainly related to DNA polymerase activity, DNA replication, and nucleotidyltransferase activity. In addition, entries for reverse transcription and endonuclease activity were enriched, indicating that ZGA interfered under HT conditions. Further comparison of the experimental results with the porcine ZGA gene revealed 39 ZGA genes among the DEGs. KEGG and GSEA analysis showed that the oxidative phosphorylation pathway was significantly enriched and signaling pathways related to energy metabolism were significantly downregulated. We also found that NDUFA6 and CDKN1A were located at the center of the protein-protein interaction network diagram of the DEGs. In summary, HT conditions affect mitochondrial function and oxidative phosphorylation levels, and lead to changes in the expression pattern of ZGA in early porcine embryos, with its hub genes NDUFA6 and CDKN1A., (© 2024. The Author(s).)

Dynamic changes in maternal‒zygotic transition (MZT) require complex regulation of zygote formation, maternal transcript decay, embryonic genome activation (EGA), and cell cycle progression. Although these changes are well described, some key regulatory factors are still elusive. Sirtuin-1 (SIRT1), an NAD + -dependent histone deacetylase, is a versatile driver of MZT via its epigenetic and nonepigenetic substrates. This study focused on the dynamics of SIRT1 in early embryos and its contribution to MZT. A conditional SIRT1-deficient knockout mouse model was used, accompanied by porcine and human embryos. Embryos across mammalian species showed the prominent localization of SIRT1 in the nucleus throughout early embryonic development. Accordingly, SIRT1 interacts with histone H4 on lysine K16 (H4K16) in both mouse and human blastocysts. While maternal SIRT1 is dispensable for MZT, at least one allele of embryonic Sirt1 is required for early embryonic development around the time of EGA. This role of SIRT1 is surprisingly mediated via a transcription-independent mode of action., (© 2024. The Author(s).)

Previous research has highlighted significant phenotypic discrepancies between knockout and knockdown approaches in zebrafish, raising concerns about the reliability of these methods. However, our study suggests that these differences are not as pronounced as was once believed. By carefully examining the roles of maternal and zygotic gene contributions, we demonstrate that these factors significantly influence phenotypic outcomes, often accounting for the observed discrepancies. Our findings emphasize that morpholinos, despite their potential off-target effects, can be effective tools when used with rigorous controls. We introduce the concept of graded maternal contribution, which explains how the uneven distribution of maternal mRNA and proteins during gametogenesis impacts phenotypic variability. Our research categorizes genes into three types-susceptible, immune, and "Schrödinger" (conditional)-based on their phenotypic expression and interaction with genetic compensation mechanisms. This distinction provides new insights into the paradoxical outcomes observed in genetic studies. Ultimately, our work underscores the importance of considering both maternal and zygotic contributions, alongside rigorous experimental controls, to accurately interpret gene function and the mechanisms underlying disease. This study advocates for the continued use of morpholinos in conjunction with advanced genetic tools like CRISPR/Cas9, stressing the need for a meticulous experimental design to optimize the utility of zebrafish in genetic research and therapeutic development.

Grapevine (Vitis vinifera L.) crops are continuously exposed to biotic and abiotic stresses, which can cause genetic and epigenetic alterations. To determine the possible effects of grapevine cryopreservation on the regulation of DNA demethylase genes, this work studied the expression of DNA demethylase genes in cryopreserved and post-cryopreserved grapevine tissues. V. vinifera DNA demethylases were characterized by in silico analysis, and gene expression quantification was conducted by RT‒qPCR. Three DNA demethylase sequences were found: VIT_13s0074g00450 (VvDMT), VIT_08s0007g03920 (VvROS1), and VIT_06s0061g01270 (VvDML3). Phylogenetic analysis revealed that the sequences from V. vinifera and A. thaliana had a common ancestry. In the promoters of responsive elements to transcription factors such as AP-2, Myb, bZIP, TBP, and GATA, the conserved domains RRM DME and Perm CXXC were detected. These responsive elements play roles in the response to abiotic stress and the regulation of cell growth. These data helped us characterize the V. vinifera DNA demethylase genes. Gene expression analysis indicated that plant vitrification solution 2 (PVS2) treatment does not alter the expression of DNA demethylase genes. The expression levels of VvDMT and VvROS1 increased in response to cryopreservation by vitrification. Furthermore, in post-cryopreservation, VvROS1 was highly induced, and VvDML3 was repressed in all the treatment groups. Gene expression differences between different treatments and tissues may play roles in controlling methylation patterns during gene regulation in tissues stressed by cryopreservation procedures and in the post-cryopreservation period during plant growth and development., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Major zygotic genome activation (ZGA) occurs at the late 2-cell stage and involves the activation of thousands of genes, supporting early embryonic development. The reasons underlying the regulation of ZGA are not clear. Acetylation modifications of histone tails promote transcriptional activation, and the maternal deletion of H4K16ac leads to failure in ZGA. GATAD2B is one of the core subunits of the nucleosome remodelling and histone deacetylation (NuRD) complex. Our research has shown that GATAD2B exhibits specific nucleus localization and high protein expression from the late 2-cell stage to the 8-cell stage. This intriguing phenomenon prompted us to investigate the relationship between GATAD2B and the ZGA. We discovered a distinctive pattern of GATAD2B, starting from the late 2-cell stage with nuclear localization. GATAD2B depletion resulted in defective embryonic development, including increased DNA damage at morula, decreased blastocyst formation rate, and abnormal differentiation of ICM/TE lineages. Consistent with the delay during the cleavage stage, the transcriptome analysis of the 2-cell embryo revealed inhibition of the cell cycle G2/M phase transition pathway. Furthermore, the GATAD2B proteomic data provided clear evidence of a certain association between GATAD2B and molecules involved in the cell cycle pathway. As hypothesized, GATAD2B-deficient 2-cell embryos exhibited abnormalities in ZGA during the maternal-to-embryonic transition, with lower expression of the major ZGA marker MERVL. Overall, our results demonstrate that GATAD2B is essential for early embryonic development, in part through facilitating ZGA., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In mammals, global passive demethylation contributes to epigenetic reprogramming during early embryonic development. At this stage, the majority of DNA-methyltransferase 1 (DNMT1) protein is excluded from nucleus, which is considered the primary cause. However, whether the remaining nuclear activity of DNMT1 is regulated by additional mechanisms is unclear. Here, we report that nuclear DNMT1 abundance is finetuned through proteasomal degradation in mouse zygotes. We identify a maternal factor, Pramel15, which targets DNMT1 for degradation via Cullin-RING E3 ligases. Loss of Pramel15 elevates DNMT1 levels in the zygote pronuclei, impairs zygotic DNA demethylation, and causes a stochastic gain of DNA methylation in early embryos. Thus, Pramel15 can modulate the residual level of DNMT1 in the nucleus during zygotic DNA replication, thereby ensuring efficient DNA methylation reprogramming in early embryos., (© 2024. The Author(s).)

The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN -modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.

The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species., Competing Interests: Declaration of interests K.Y. and T.M. are cofounders of C4U Corporation. T.M. is an outside board member of and K.Y. and K.T. are scientific advisors to C4U., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The antidepressant venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is commonly prescribed to treat major depressive disorder and is found at high concentrations in the aquatic environment. Concerns have been raised related to the health of aquatic organisms in response to this nontargeted pharmaceutical exposure. For instance, we previously demonstrated that exposure to venlafaxine perturbs neurodevelopment, leading to behavioural alterations in zebrafish (Danio rerio). We also observed disruption in serotonin expression in the pineal and raphe, regions critical in regulating circadian rhythms, leading us to hypothesize that zygotic exposure to venlafaxine disrupts the circadian locomotor rhythm in larval zebrafish. To test this, we microinjected zebrafish embryos with venlafaxine (1 or 10 ng) and recorded the locomotor activity in 5-day-old larvae over a 24-h period. Venlafaxine deposition reduced larval locomotor activity during the light phase, but not during the dark phase of the diurnal cycle. The melatonin levels were higher in the dark compared to during the light photoperiod and this was not affected by embryonic venlafaxine deposition. Venlafaxine exposure also did not affect the transcript abundance of clock genes, including clock1a, bmal2, cry1a and per2, which showed a clear day/night rhythmicity. A notable finding was that exposure to luzindole, a melatonin receptor antagonist, decreased the locomotor activity in the control group in light, whereas the activity was higher in larvae raised from the venlafaxine-deposited embryos. Overall, zygotic exposure to venlafaxine disrupts the locomotor activity of larval zebrafish fish during the day, demonstrating the capacity of antidepressants to disrupt the circadian rhythms in behaviour. Our results suggest that disruption in melatonin signalling may be playing a role in the venlafaxine impact on circadian behaviour, but further investigation is required to elucidate the possible mechanisms in larval zebrafish., (© 2024 The Author(s). Journal of Pineal Research published by John Wiley & Sons Ltd.)

Study Question: Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes?, Summary Answer: Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes., What Is Known Already: Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis., Study Design, Size, Duration: This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months., Participants/materials, Setting, Methods: All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling., Main Results and the Role of Chance: Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining., Large Scale Data: The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633)., Limitations, Reasons for Caution: It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation., Wider Implications of the Findings: Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation., Study Funding/competing Interest(s): This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Specific cell depletion is a common means to study the physiological function of cell lineages and tissue regeneration. However, 100% depletion is difficult to achieve with existing cell depletion strategies. With the increasing maturity of CRISPR/Cas9 technology, it is increasingly used for the depletion of various cells. However, even with this technology, it is difficult to complete the depletion of specific gene knockout cells. For this reason, cell depletion with the use of repetitive sequences as the target of CRISPR/Cas9 was explored using zebrafish. All cells were used as the target cells for the first set of experiments. The results showed that injection of a mixture of DANA-gRNA and Cas9 mRNA into zygotes resulted in substantial cell apoptosis. Cells are almost invisible in the embryonic animal pole during the dome stage. The activities of the caspase-3 and caspase-9 proteins and the mRNA level of the P53 gene were significantly increased. Then, primordial germ cells (PGCs) in embryos were used as the target cells in subsequent experiments. To specifically knock out PGCs, we injected the mix of DANA-gRNA, pkop: Cas9 plasmid (the kop promotor allows Cas9 expression only in PGCs), and eGFP-nos3'UTR mRNA into zebrafish fertilized eggs. The results revealed that the activity of the caspase-3 protein was significantly increased, and the mRNA levels of P53, ku70, and ku80 were significantly upregulated, while the number of PGCs decreased gradually. Few PGCs labeled with GFP could be seen 20 h post-fertilization (hpf), and no PGCs could be seen at the germinal ridge 24 hpf. Therefore, the combination of CRISPR/Cas9 technology and repetitive sequences can achieve efficient cell depletion regardless of whether there is generalized expression or expression in specific cells. These results indicate that it is feasible to eliminate cells by using repeat sequences as CRISPR/Cas9 system target sites., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency., (© 2024. The Society for In Vitro Biology.)

Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs., (© 2024. The Society for In Vitro Biology.)

Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used., (© 2024. The Society for In Vitro Biology.)

Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans . If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans , we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes., Competing Interests: EB, CB, MM, SQ, KM, FM No competing interests declared, (© 2024, Beath et al.)

The first embryonic division represents a starting point for the development of a new individual. In many species, tight control over the first embryonic division ensures its accuracy. However, the first division in humans is often erroneous and can impair embryo development. To delineate the spatiotemporal organization of the first mitotic division typical for normal human embryo development, we systematically analyzed a unique timelapse dataset of 300 IVF embryos that developed into healthy newborns. The zygotic division pattern of these best-quality embryos was compared to their siblings that failed to implant or arrested during cleavage stage. We show that division at the right angle to the juxtaposed pronuclei is preferential and supports faithful zygotic division. Alternative configurations of the first mitosis are associated with reduced clustering of nucleoli and multinucleation at the 2-cell stage, which are more common in women of advanced age. Collectively, these data imply that orientation of the first division predisposes human embryos to genetic (in)stability and may contribute to aneuploidy and age-related infertility., (© 2024. The Author(s).)

The timing of DNA replication in mammals is crucial for minimizing errors and influenced by genome usage and chromatin states. Replication timing in the newly formed mammalian embryo remains poorly understood. Here, we have investigated replication timing in mouse zygotes and 2-cell embryos, revealing that zygotes lack a conventional replication timing program, which then emerges in 2-cell embryos. This program differs from embryonic stem cells and generally correlates with transcription and genome compartmentalization of both parental genomes. However, consistent and systematic differences existed between the replication timing of the two parental genomes, including considerably later replication of maternal pericentromeric regions compared to paternal counterparts. Moreover, maternal chromatin modified by Polycomb Repressive Complexes in the oocyte, undergoes early replication, despite belonging to the typically late-replicating B-compartment of the genome. This atypical and asynchronous replication of the two parental genomes may advance our understanding of replication stress in early human embryos and trigger strategies to reduce errors and aneuploidies., (© 2024. The Author(s).)

While most dizygotic twins have a dichorionic placenta, rare cases of dizygotic twins with a monochorionic placenta have been reported. The monochorionic placenta in dizygotic twins allows in utero exchange of embryonic cells, resulting in chimerism in the twins. In practice, this chimerism is incidentally identified in mixed ABO blood types or in the presence of cells with a discordant sex chromosome. Here, we applied whole-genome sequencing to one triplet and one twin family to precisely understand their zygotic compositions, using millions of genomic variants as barcodes of zygotic origins. Peripheral blood showed asymmetrical contributions from two sister zygotes, where one of the zygotes was the major clone in both twins. Single-cell RNA sequencing of peripheral blood tissues further showed differential contributions from the two sister zygotes across blood cell types. In contrast, buccal tissues were pure in genetic composition, suggesting that in utero cellular exchanges were confined to the blood tissues. Our study illustrates the cellular history of twinning during human development, which is critical for managing the health of chimeric individuals in the era of genomic medicine., Competing Interests: Declaration of interests Y.S.J. is the co-founder and chief genomics officer of Inocras, Inc. J.S.L. is the co-founder and chief innovation officer of Inocras, Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Malaria, a global health concern, is caused by parasites of the Plasmodium genus, which undergo gametogenesis in the midgut of mosquitoes after ingestion of an infected blood meal. The resulting male and female gametes fuse to form a zygote, which differentiates into a motile ookinete. After traversing the midgut epithelium, the ookinete differentiates into an oocyst on the basal side of the epithelium., Methods: Membrane proteins with increased gene expression levels from the gamete to oocyst stages in P. berghei were investigated utilizing PlasmoDB, the functional genomic database for Plasmodium spp. Based on this analysis, we selected the 184-kDa membrane protein, Pb184, for further study. The expression of Pb184 was further confirmed through immunofluorescence staining, following which we examined whether Pb184 is involved in fertilization using antibodies targeting the C-terminal region of Pb184 and biotin-labeled C-terminal region peptides of Pb184., Results: Pb184 is expressed on the surface of male and female gametes. The antibody inhibited zygote and ookinete formation in vitro. When mosquitoes were fed on parasite-infected blood containing the antibody, oocyst formation decreased on the second day after feeding. Synthesized biotin-labeled peptides matching the C-terminal region of Pb184 bound to the female gamete and the residual body of male gametes, and inhibited differentiation into ookinetes in the in vitro culture system., Conclusions: These results may be useful for the further studying the fertilization mechanism of Plasmodium protozoa. There is also the potential for their application as future tools to prevent malaria transmission., (© 2024. The Author(s).)

In spite of its essential role in culture media, the precise influence of lactate on early mouse embryonic development remains elusive. Previous studies have implicated lactate accumulation in medium affecting histone acetylation. Recent research has underscored lactate-derived histone lactylation as a novel epigenetic modification in diverse cellular processes and diseases. Our investigation demonstrated that the absence of sodium lactate in the medium resulted in a pronounced 2-cell arrest at the late G2 phase in embryos. RNA-seq analysis revealed that the absence of sodium lactate significantly impaired the maternal-to-zygotic transition (MZT), particularly in zygotic gene activation (ZGA). Investigations were conducted employing Cut&Tag assays targeting the well-studied histone acetylation and lactylation sites, H3K18la and H3K27ac, respectively. The findings revealed a noticeable reduction in H3K18la modification under lactate deficiency, and this alteration showed a significant correlation with changes in gene expression. In contrast, H3K27ac exhibited minimal correlation. These results suggest that lactate may preferentially influence early embryonic development through H3K18la rather than H3K27ac modifications., (© 2024. The Author(s).)

Microinjection of CRISPR/Cas9 requires the availability of zygotes that implies animal breeding, superovulation schemes, and embryo collection. Vitrification of zygotes may allow having ready-to-use embryos and to temporally dissociate the workload of embryo production from microinjection. In this study, fresh (F group) or vitrified (V group) zygotes were microinjected with CRISPR/Cas9 system to test the hypothesis that vitrified zygotes could be a suitable source of embryos for microinjection. In Experiment 1 (in vitro evaluation), B6D2F1/J zygotes were microinjected and cultured until blastocyst stage. Embryo survival and cleavage rates after microinjection were similar between groups (~50% and ~80% respectively; P = NS). Development rate was significantly higher for F than V group (55.0% vs. 32.6%, respectively; P<0.05). Mutation rate did not show statistical differences among groups (P = NS). In Experiment 2 (in vivo evaluation), C57BL/6J zygotes were microinjected and transferred to recipient females. Embryo survival was significantly lower in fresh than in vitrified zygotes (49.2% vs. 62.7%, respectively; P<0.05). Cleavage rate did not show statistical differences (~70%; P = NS). Pregnancy rate (70.0% vs. 58.3%) and birth rate (11.9% vs. 11.2%) were not different between groups (F vs. V group; P = NS). Offspring mutation rate was higher for F than V group, in both heterodimer analysis (73.7% vs. 33.3%, respectively; P = 0.015) and Sanger sequencing (89.5% vs. 41.7%, respectively; P = 0.006). In conclusion, vitrified-warmed zygotes present a viable alternative source for CRISPR/Cas9 microinjection when the production of fresh embryos is impeded by limited technical support. The possibility of zygote cryobanking to perform microinjection sessions on demand seems to be a suitable alternative to avoid the breeding and maintenance of animals all over the year, enhancing the implementation of CRISPR technology., Competing Interests: NO authors have competing interests, (Copyright: © 2024 Schlapp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Long interspersed nuclear element-1 (LINE-1 or L1) is a retrotransposon group that constitutes 17% of the human genome and shows variable expression across cell types. However, the control of L1 expression and its function in gene regulation are incompletely understood. Here we show that L1 transcription activates long-range gene expression. Genome-wide CRISPR-Cas9 screening using a reporter driven by the L1 5' UTR in human cells identifies functionally diverse genes affecting L1 expression. Unexpectedly, altering L1 expression by knockout of regulatory genes impacts distant gene expression. L1s can physically contact their distal target genes, with these interactions becoming stronger upon L1 activation and weaker when L1 is silenced. Remarkably, L1s contact and activate genes essential for zygotic genome activation (ZGA), and L1 knockdown impairs ZGA, leading to developmental arrest in mouse embryos. These results characterize the regulation and function of L1 in long-range gene activation and reveal its importance in mammalian ZGA., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

During human embryonic development, early cleavage-stage embryos are more susceptible to errors. Studies have shown that many problems occur during the first mitosis, such as direct cleavage, chromosome segregation errors, and multinucleation. However, the mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, in the present study, we image discarded living human two-pronuclear stage zygotes using fluorescent labeling and confocal microscopy without microinjection of DNA or mRNA and investigate the association between spindle shape and nuclear abnormality during the first mitosis. We observe that the first mitotic spindles vary, and low-aspect-ratio-shaped spindles tend to lead to the formation of multiple nuclei at the 2-cell stage. Moreover, we observe defocusing poles in many of the first mitotic spindles, which are strongly associated with multinucleation. Additionally, we show that differences in the positions of the centrosomes cause spindle abnormality in the first mitosis. Furthermore, many multinuclei are modified to form mononuclei after the second mitosis because the occurrence of pole defocusing is firmly reduced. Our study will contribute markedly to research on the occurrence of mitotic errors during the early cleavage of human embryos., (© 2024. The Author(s).)

In mice, transcription from the zygotic genome is initiated at the mid-one-cell stage, and occurs promiscuously in many areas of the genome, including intergenic regions. Regulated transcription from selected genes is established during the two-cell stage. This dramatic change in the gene expression pattern marks the initiation of the gene expression program and is essential for early development. We investigated the involvement of the histone variants H3.1/3.2 in the regulation of changes in gene expression pattern during the two-cell stage. Immunocytochemistry analysis showed low nuclear deposition of H3.1/3.2 in the one-cell stage, followed by a rapid increase in the late two-cell stage. Where chromatin structure is normally closed between the one- and two-cell stages, it remained open until the late two-cell stage when H3.1/3.2 were knocked down by small interfering RNA. Hi-C analysis showed that the formation of the topologically associating domain was disrupted in H3.1/3.2 knockdown (KD) embryos. Promiscuous transcription was also maintained in the late two-cell stage in H3.1/3.2 KD embryos. These results demonstrate that H3.1/3.2 are involved in the initial process of the gene expression program after fertilization, through the formation of a closed chromatin structure to execute regulated gene expression during the two-cell stage., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Once fertilized, mouse zygotes rapidly proceed to zygotic genome activation (ZGA), during which long terminal repeats (LTRs) of murine endogenous retroviruses with leucine tRNA primer (MERVL) are activated by a conserved homeodomain-containing transcription factor, DUX. However, Dux -knockout embryos produce fertile mice, suggesting that ZGA is redundantly driven by an unknown factor(s). Here, we present multiple lines of evidence that the multicopy homeobox gene, Obox4 , encodes a transcription factor that is highly expressed in mouse two-cell embryos and redundantly drives ZGA. Genome-wide profiling revealed that OBOX4 specifically binds and activates MERVL LTRs as well as a subset of murine endogenous retroviruses with lysine tRNA primer (MERVK) LTRs. Depletion of Obox4 is tolerated by embryogenesis, whereas concomitant Obox4 / Dux depletion markedly compromises embryonic development. Our study identified OBOX4 as a transcription factor that provides genetic redundancy to preimplantation development., Competing Interests: YG, TK, KI, KM, MH, TL, AS, HI, NO, SM, MS, AO, HS No competing interests declared, (© 2024, Guo, Kitano, Inoue et al.)

Precise integration of DNA constructs greater than 3 kb into mouse zygotes is difficult. Here, we present a protocol for large DNA transgenesis in mice using the Cas9+Bxb1 toolbox. We describe steps for choosing mouse strains with preplaced attachment sites. We then detail procedures for microinjecting mouse zygotes with the plasmid donor DNA construct to generate transgenic mice by recombination-mediated cassette exchange. This protocol has the potential for application in exploring the functional implications of large structural variations in cancer. For complete details on the use and execution of this protocol, please refer to Low et al. 1 and Hosur et al. 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Zygotic genome activation (ZGA) is a pivotal event in mammalian embryogenesis, marking the transition from maternal to zygotic control of development. During the ZGA process that is characterized by the intricate cascade of gene expression, who tipped the first domino in a meticulously arranged sequence is a subject of paramount interest. Recently, Dux, Obox and Nr5a2 were identified as pioneer transcription factors that reside at the top of transcriptional hierarchy. Through co-option of retrotransposon elements as hubs for transcriptional activation, these pioneer transcription factors rewire the gene regulatory network, thus initiating ZGA. In this review, we provide a snapshot of the mechanisms underlying the functions of these pioneer transcription factors. We propose that ZGA is the starting point where the embryo's own genome begins to influence development trajectory, therefore in-depth dissecting the functions of pioneer transcription factors during ZGA will form a cornerstone of our understanding for early embryonic development, which will pave the way for advancing our grasp of mammalian developmental biology and optimizing in vitro production (IVP) techniques.

Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems., Competing Interests: Competing interests statement:The authors declare no competing interest.

Plants are dependent on divisions of stem cells to establish cell lineages required for growth. During embryogenesis, early division products are considered to be stem cells, whereas during post-embryonic development, stem cells are present in meristems at the root and shoot apex. PLETHORA/AINTEGUMENTA-LIKE (PLT/AIL) transcription factors are regulators of post-embryonic meristem function and are required to maintain stem cell pools. Despite the parallels between embryonic and post-embryonic stem cells, the role of PLTs during early embryogenesis has not been thoroughly investigated. Here, we demonstrate that the PLT regulome in the zygote, and apical and basal cells is in strong congruence with that of post-embryonic meristematic cells. We reveal that out of all six PLTs, only PLT2 and PLT4/BABY BOOM (BBM) are expressed in the zygote, and that these two factors are essential for progression of embryogenesis beyond the zygote stage and first divisions. Finally, we show that other PLTs can rescue plt2 bbm defects when expressed from the PLT2 and BBM promoters, establishing upstream regulation as a key factor in early embryogenesis. Our data indicate that generic PLT factors facilitate early embryo development in Arabidopsis by induction of meristematic potential., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce experimentally. Here, we report the first success in establishing animal models for CCRs. Mutation in Recql5, a crucial member of the DNA helicase RecQ family involved in DNA replication, transcription, and repair, enabled CRISPR/Cas9-mediated CCRs, establishing a mouse model containing triple fusion genes and megabase-sized inversions. Some of these structural features of individual chromosomal rearrangements use template switching and microhomology-mediated break-induced replication mechanisms and are reminiscent of the newly described phenomenon "chromoanasynthesis." These data show that Recql5 mutant mice could be a powerful tool to analyze the pathogenesis of CCRs (particularly chromoanasynthesis) whose underlying mechanisms are poorly understood. The Recql5 mutants generated in this study are to be deposited at key animal research facilities, thereby making them accessible for future research on CCRs., Competing Interests: Conflicts of interest. The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Rapid cleavage divisions and the transition from maternal to zygotic control of gene expression are the hallmarks of early embryonic development in most species. Early development in insects, fish and amphibians is characterized by several short cell cycles with no gap phases, necessary for the rapid production of cells prior to patterning and morphogenesis. Maternal mRNAs and proteins loaded into the egg during oogenesis are essential to drive these rapid early divisions. Once the function of these maternal inputs is complete, the maternal-to-zygotic transition (MZT) marks the handover of developmental control to the gene products synthesized from the zygotic genome. The MZT requires three major events: the removal of a subset of maternal mRNAs, the initiation of zygotic transcription, and the remodeling of the cell cycle. In each species, the MZT occurs at a highly reproducible time during development due to a series of feedback mechanisms that tightly couple these three processes. Dissecting these feedback mechanisms and their spatiotemporal control will be essential to understanding the control of the MZT. In this primer, we outline the mechanisms that govern the major events of the MZT across species and highlight the role of feedback mechanisms that ensure the MZT is precisely timed and orchestrated., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nicotiana attenuata styles preferentially select pollen from among accessions with corresponding expression patterns of NaS-like-RNases (SLRs), and the postpollination ethylene burst (PPEB) is an accurate predictor of seed siring success. However, the ecological consequences of mate selection, its effect on the progeny, and the role of SLRs in the control of ethylene signaling remain unknown. We explored the link between the magnitude of the ethylene burst and expression of the SLRs in a set of recombinant inbred lines (RILs), dissected the genetic underpinnings of mate selection through genome-wide association study (GWAS), and examined its outcome for phenotypes in the next generation. We found that high levels of PPEB are associated with the absence of SLR2 in most of the tested RILs. We identified candidate genes potentially involved in the control of mate selection and showed that pollination of maternal genotypes with their favored pollen donors produces offspring with longer roots. When the maternal genotypes are only able to select against nonfavored pollen donors, the selection for such positive traits is abolished. We conclude that plants' ability of mate choice contributes to measurable changes in progeny phenotypes and is thus likely a target of selection., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

Adaptive response to physiological oxygen levels (physO 2 ; 5% O 2 ) enables embryonic survival in a low-oxygen developmental environment. However, the mechanism underlying the role of physO 2 in supporting preimplantation development, remains elusive. Here, we systematically studied oxygen responses of hallmark events in preimplantation development. Focusing on impeded transcriptional upregulation under atmospheric oxygen levels (atmosO 2 ; 20% O 2 ) during the 2-cell stage, we functionally identified a novel role of HIF-1α in promoting major zygotic genome activation by serving as an oxygen-sensitive transcription factor. Moreover, during blastocyst formation, atmosO 2 impeded H3K4me3 and H3K27me3 deposition by deregulating histone-lysine methyltransferases, thus impairing X-chromosome inactivation in blastocysts. In addition, we found atmosO 2 impedes metabolic shift to glycolysis before blastocyst formation, thus resulting a low-level histone lactylation deposition. Notably, we also reported an increased sex-dimorphic oxygen response of embryos upon preimplantation development. Together, focusing on genetic and epigenetic events that are essential for embryonic survival and development, the present study advances current knowledge of embryonic adaptive responses to physO 2 , and provides novel insight into mechanism underlying irreversibly impaired developmental potential due to a short-term atmosO 2 exposure., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Totipotency refers to the ability of a single cell to give rise to all the different cell types in the body. Terminally differentiated germ cells (sperm and oocytes) undergo reprogramming, which results in the acquisition of totipotency in zygotes. Since the 1990s, numerous studies have focused on the mechanisms of totipotency. With the emergence of the concept of epigenetic reprogramming, which is important for the undifferentiated and differentiated states of cells, the epigenomes of germ cells and fertilized oocytes have been thoroughly analyzed. However, in early immunostaining studies, detailed epigenomic information was difficult to obtain. In recent years, the explosive development of next-generation sequencing has made it possible to acquire genome-wide information and the rise of genome editing has facilitated the analysis of knockout mice, which was previously difficult. In addition, live imaging can effectively analyze zygotes and 2-cell embryos, for which the number of samples is limited, and provides biological insights that cannot be obtained by other methods. In this review, the progress of our research using these advanced techniques is traced back from the present to its earliest years.

The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3-deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3, oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes., (Copyright © 2024. Published by Elsevier B.V.)