RESUMEN

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.

Asunto(s)

Desarrollo Embrionario , Ratones Noqueados , Sirtuina 1 , Cigoto , Sirtuina 1/metabolismo , Sirtuina 1/genética , Animales , Cigoto/metabolismo , Humanos , Ratones , Desarrollo Embrionario/genética , Femenino , Histonas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Blastocisto/metabolismo , Porcinos , Embrión de Mamíferos/metabolismo

RESUMEN

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.

Asunto(s)

Sistemas CRISPR-Cas , Electroporación , Fertilización In Vitro , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Ribonucleoproteínas , Cigoto , Animales , Edición Génica/métodos , Electroporación/métodos , Cigoto/metabolismo , Fertilización In Vitro/métodos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Ovinos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Miostatina/genética , Femenino , Animales Modificados Genéticamente

RESUMEN

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.

Asunto(s)

Búfalos , Sistemas CRISPR-Cas , Electroporación , Edición Génica , Técnicas de Transferencia Nuclear , Transfección , Animales , Búfalos/genética , Electroporación/veterinaria , Electroporación/métodos , Femenino , Embarazo , Edición Génica/métodos , Edición Génica/veterinaria , Transfección/veterinaria , Transfección/métodos , Técnicas de Transferencia Nuclear/veterinaria , Miostatina/genética , Cigoto/metabolismo

RESUMEN

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.

Asunto(s)

Núcleo Celular , ADN (Citosina-5-)-Metiltransferasa 1 , Desmetilación del ADN , Cigoto , Animales , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , Cigoto/metabolismo , Ratones , Núcleo Celular/metabolismo , Femenino , Metilación de ADN , Proteolisis , Desarrollo Embrionario/genética , Masculino , Embrión de Mamíferos/metabolismo , Ratones Noqueados , Regulación del Desarrollo de la Expresión Génica , Replicación del ADN

RESUMEN

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.

Asunto(s)

Sistemas CRISPR-Cas , Edición Génica , Cigoto , Animales , Cigoto/metabolismo , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Ratas , Ratones , Femenino

RESUMEN

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.

Asunto(s)

Criopreservación , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Vitis , Vitis/genética , Vitis/crecimiento & desarrollo , Criopreservación/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Filogenia , Semillas/genética , Semillas/crecimiento & desarrollo , Desmetilación del ADN , Cigoto/metabolismo , Metilación de ADN , Crioprotectores/farmacología

RESUMEN

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.

Asunto(s)

Histonas , Ácido Láctico , Cigoto , Histonas/metabolismo , Histonas/genética , Animales , Acetilación , Cigoto/metabolismo , Ratones , Ácido Láctico/metabolismo , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Epigénesis Genética , Genoma , Procesamiento Proteico-Postraduccional

RESUMEN

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.

Asunto(s)

Caenorhabditis elegans , Katanina , Cinesinas , Cigoto , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Katanina/metabolismo , Katanina/genética , Cigoto/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Masculino , Ataxina-2/genética , Ataxina-2/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Espermatozoides/metabolismo , Femenino , Fertilización

RESUMEN

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.

Asunto(s)

Sistemas CRISPR-Cas , Microinyecciones , Cigoto , Animales , Cigoto/metabolismo , Femenino , Ratones , Criopreservación/métodos , Embarazo , Ratones Endogámicos C57BL , Transferencia de Embrión/métodos , Masculino , Vitrificación , Desarrollo Embrionario/genética

RESUMEN

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.

Asunto(s)

Núcleo Celular , Desarrollo Embrionario , Mitosis , Huso Acromático , Cigoto , Humanos , Huso Acromático/metabolismo , Femenino , Núcleo Celular/metabolismo , Cigoto/metabolismo , Cigoto/citología , Fertilización In Vitro , Embrión de Mamíferos/citología , Fase de Segmentación del Huevo/citología , Masculino

RESUMEN

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.

Asunto(s)

Replicación del ADN , Embrión de Mamíferos , Cigoto , Animales , Femenino , Ratones , Cigoto/metabolismo , Masculino , Embrión de Mamíferos/metabolismo , Cromatina/metabolismo , Cromatina/genética , Oocitos/metabolismo , Momento de Replicación del ADN , Genoma , Desarrollo Embrionario/genética , Ratones Endogámicos C57BL

RESUMEN

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.

Asunto(s)

Fertilización , Plasmodium berghei , Proteínas Protozoarias , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Animales , Femenino , Masculino , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Ratones , Células Germinativas/metabolismo , Malaria/parasitología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Cigoto/metabolismo , Anopheles/parasitología , Anopheles/metabolismo , Oocistos/metabolismo , Gametogénesis/genética

RESUMEN

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.

Asunto(s)

Sistemas CRISPR-Cas , Elementos de Nucleótido Esparcido Largo , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Animales , Ratones , Activación Transcripcional , Cigoto/metabolismo , Transcripción Genética , Regulación de la Expresión Génica , Regiones no Traducidas 5'

RESUMEN

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.

Asunto(s)

Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Histonas , Cigoto , Animales , Ratones , Cigoto/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/efectos de los fármacos , Histonas/metabolismo , Femenino , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Procesamiento Proteico-Postraduccional , Código de Histonas , Embrión de Mamíferos/metabolismo , Ubiquitinación , Ciclopentanos , Pirimidinas

RESUMEN

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.

Asunto(s)

Ritmo Circadiano , Larva , Locomoción , Clorhidrato de Venlafaxina , Pez Cebra , Animales , Pez Cebra/embriología , Clorhidrato de Venlafaxina/farmacología , Clorhidrato de Venlafaxina/toxicidad , Larva/efectos de los fármacos , Locomoción/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Cigoto/efectos de los fármacos , Cigoto/metabolismo , Actividad Motora/efectos de los fármacos , Melatonina/farmacología

RESUMEN

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.

Asunto(s)

Genoma , Factores de Transcripción , Cigoto , Cigoto/metabolismo , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Humanos , Regulación del Desarrollo de la Expresión Génica , Desarrollo Embrionario/genética , Retroelementos/genética , Activación Transcripcional/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo

RESUMEN

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.

Asunto(s)

Diferenciación Celular , Empalmosomas , Animales , Humanos , Ratones , Blastocisto/metabolismo , Blastocisto/citología , Blastómeros/metabolismo , Blastómeros/citología , Reprogramación Celular , Desarrollo Embrionario/genética , Estratos Germinativos/metabolismo , Estratos Germinativos/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Empalme del ARN , Empalmosomas/metabolismo , Células Madre Totipotentes/metabolismo , Células Madre Totipotentes/citología , Cigoto/metabolismo , Células Cultivadas , Modelos Moleculares , Estructura Terciaria de Proteína , Genoma Humano , Análisis de la Célula Individual , Factor 15 de Diferenciación de Crecimiento/química , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/metabolismo , Epigenómica , Linaje de la Célula

RESUMEN

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.

Asunto(s)

Núcleo Celular , Mitosis , Huso Acromático , Humanos , Huso Acromático/metabolismo , Núcleo Celular/metabolismo , Cigoto/citología , Cigoto/metabolismo , Embrión de Mamíferos/citología , Microscopía Confocal , Centrosoma/metabolismo , Desarrollo Embrionario/fisiología , Femenino

RESUMEN

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.

Asunto(s)

Cigoto , Cigoto/metabolismo , Cigoto/crecimiento & desarrollo , Animales , Regulación del Desarrollo de la Expresión Génica , Desarrollo Embrionario , Femenino , ARN Mensajero Almacenado/metabolismo , ARN Mensajero Almacenado/genética

RESUMEN

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.

Asunto(s)

Proteínas de Homeodominio , Cigoto , Animales , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Cigoto/metabolismo , Ratones , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Genoma , Ratones Noqueados