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1.
BMC Res Notes ; 14(1): 389, 2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34627381

RESUMO

OBJECTIVE: Lipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection. RESULTS: Zona pellucida (ZP)-free zygotes collected at 5, 10, and 15 h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA) targeting GGTA1, and Cas9 for 5 h. Lipofection of zygotes collected at 10 and 15 h from the start of IVF yielded mutant blastocysts. Next, ZP-free zygotes collected at 10 h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas9 for 2.5, 5, 10, or 20 h. The blastocyst formation rate of zygotes treated for 20 h was significantly lower (p < 0.05) than those of the other groups, and no mutant blastocysts were obtained. Moreover, the mutation rates of the resulting blastocysts decreased as the incubation time increased. In conclusion, a lipofection-mediated gene editing system using the CRISPR/Cas9 system in ZP-zygotes is feasible; however, further improvements in the gene editing efficiency are required.


Assuntos
Edição de Genes , Zigoto , Animais , Blastocisto , Sistemas CRISPR-Cas/genética , RNA Guia , Suínos
3.
Elife ; 102021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34342574

RESUMO

During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, we hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as chromatin-linked adaptor for male-specific lethal (MSL) proteins (CLAMP). Here, we demonstrate that Drosophila embryonic development requires maternal CLAMP to (1) activate zygotic transcription; (2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; and (3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor that plays a targeted yet essential role in ZGA.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genoma de Inseto , Proteínas Nucleares/genética , Ativação Transcricional , Animais , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Zigoto/metabolismo
4.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445470

RESUMO

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory "aged" oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.


Assuntos
Núcleo Celular/patologia , Citoplasma/patologia , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário , Fertilização In Vitro/métodos , Zigoto/patologia , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Zigoto/metabolismo
5.
Mar Environ Res ; 170: 105453, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34425401

RESUMO

The macro- and microalgae have been found to inhibit the growth and photosynthesis of one another due to allelopathic interactions between them. Sargassum fusiformis is a common and commercially cultivated seaweed in coastal waters of the East China Sea (ECS) and usually encounters dense harmful algal blooms (HABs) formed by dinoflagellates during their sexual reproduction period. In the present study, the effects of Prorocentrum donghaiense lipophilic extracted allelochemicals on the growth and photosynthesis of S. fusiformis zygotes were probed by fast chlorophyll fluorescence rise kinetics and chlorophyll a transient analysis (JIP-test). It was found that exposure to the allelochemicals led to decreased chlorophyll a content and photosynthetic rates of the zygotes in comparison to the ones in the control. In addition, using the JIP-test, it was found that the inhibitory effects of allelochemicals on photosynthesis of the zygotes were mainly exerted on the electron transport within PSII. The decrease of photosynthetic parameters such as VJ, Mo, ϕPo, ϕo, ϕEo, PI, PTR, PET in the zygotes exposed to the allelochemicals all revealed that the obstruction of electron transport, and the dominant decrease in PET, both implied that inhibition on the dark reaction contributed to the highest photosynthetic reduction. In addition, some reaction centers (RCs) in the zygotes exposed to the allelocamicals were inactivated, which led to higher dissipation of excitation energy, as demonstrated by the significant enhancement of the photosynthetic parameter DIo/RC. All the results indicated that the lipophilic extracts contained the allelochemicals of P. donghaiense which could inhibit the growth and photosynthesis of S. fusiformis zygotes by damaging the electron acceptors and inactivating RCs, and finally block the electron transport.


Assuntos
Dinoflagelados , Sargassum , Clorofila , Clorofila A , Fluorescência , Cinética , Feromônios , Fotossíntese , Zigoto
6.
Eur J Obstet Gynecol Reprod Biol ; 264: 206-211, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34329946

RESUMO

OBJECTIVE: To explore the development and pregnancy potential of non-pronuclear (0PN) zygote-derived embryos in conventional in vitro fertilization (IVF) cycles. STUDY DESIGN: Embryonic development in 1039 oocyte retrieval cycles and clinical outcomes of 659 frozen-thawed blastocyst transfer cycles were retrospectively studied. RESULTS: Developmental potential of embryos with different blastomere numbers on day 3 were inconsistent in 0PN and 2PN groups. For 0PN-derived embryos, blastocyst rate of fast developing embryos (75.4%) was similar to that of intermediately developing embryos (72.9%), but good quality blastocyst rate of the former (49.2%) was significantly higher than that of the later (39.6%). In 2PN group, intermediately developing embryos had the highest blastocyst rate (77.9%) and good quality blastocyst rate (51.5%) (statistically significant). Comparison of frozen-thawed transfer was carried out between 0PN- and 2PN-derived blastocysts. For both single (SBT) and double blastocyst transfer (DBT) groups, no statistical differences existed between 0PN- and 2PN-derived blastocysts in clinical pregnancy rates (45.2% and 49.1% in SBT group, 64.7% and 66.4% in DBT group), implantation rates (45.2% and 49.1% in SBT group, 41.2% and 47.7% in DBT group) and live birth rates (35.5% and 36.8% in SBT group, 52.9% and 51.2% in DBT group). CONCLUSION: The developmental characteristic of 0PN-derived embryos was different from that of 2PN-derived embryos in IVF cycles. 0PN-derived blastocysts could obtain acceptable clinical pregnancy and live birth, but more studies are needed to confirm the safety..


Assuntos
Transferência Embrionária , Zigoto , Blastocisto , Feminino , Fertilização In Vitro , Humanos , Gravidez , Taxa de Gravidez , Estudos Retrospectivos
7.
Biomolecules ; 11(6)2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199637

RESUMO

Endogenous retroviruses (ERVs), previously viewed as deleterious relics of ancestral retrovirus infections, are silenced in the vast majority of cells to minimize the risk of retrotransposition. Counterintuitively, bursts of ERV transcription usually occur during maternal-to-zygotic transition (MZT) in preimplantation embryos; this is regarded as a major landmark event in the zygotic genome activation (ZGA) process, indicating that ERVs play an active part in ZGA. Evolutionarily, the interaction between ERVs and hosts is mutually beneficial. The endogenization of retrovirus sequences rewires the gene regulatory network during ZGA, and ERV repression may lower germline fitness. Unfortunately, owing to various limitations of somatic cell nuclear transfer (SCNT) technology, both developmental arrest and ZGA abnormalities occur in a high percentage of cloned embryos, accompanied by ERV silencing, which may be caused by the activation failure of upstream ERV inducers. In this review, we discuss the functions and regulation of ERVs during the ZGA process and the feasibility of temporal control over ERVs in cloned embryos via exogenous double homeobox (DUX). We hypothesize that further accurate characterization of the ERV-rewired gene regulatory network during ZGA may provide a novel perspective on the development of preimplantation embryos.


Assuntos
Clonagem de Organismos , Desenvolvimento Embrionário , Retrovirus Endógenos/metabolismo , Genoma , Técnicas de Transferência Nuclear , Zigoto/metabolismo , Animais
8.
Nat Genet ; 53(8): 1207-1220, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34267371

RESUMO

In mammalian embryos, proper zygotic genome activation (ZGA) underlies totipotent development. Double homeobox (DUX)-family factors participate in ZGA, and mouse Dux is required for forming cultured two-cell (2C)-like cells. Remarkably, in mouse embryonic stem cells, Dux is activated by the tumor suppressor p53, and Dux expression promotes differentiation into expanded-fate cell types. Long-read sequencing and assembly of the mouse Dux locus reveals its complex chromatin regulation including putative positive and negative feedback loops. We show that the p53-DUX/DUX4 regulatory axis is conserved in humans. Furthermore, we demonstrate that cells derived from patients with facioscapulohumeral muscular dystrophy (FSHD) activate human DUX4 during p53 signaling via a p53-binding site in a primate-specific subtelomeric long terminal repeat (LTR)10C element. In summary, our work shows that p53 activation convergently evolved to couple p53 to Dux/DUX4 activation in embryonic stem cells, embryos and cells from patients with FSHD, potentially uniting the developmental and disease regulation of DUX-family factors and identifying evidence-based therapeutic opportunities for FSHD.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Murinas/fisiologia , Distrofia Muscular Facioescapuloumeral/patologia , Proteína Supressora de Tumor p53/genética , Animais , Diferenciação Celular/genética , Reprogramação Celular , Dano ao DNA , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Distrofia Muscular Facioescapuloumeral/genética , Proteínas Nucleares/genética , Células-Tronco Pluripotentes/fisiologia , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/metabolismo , Zigoto/citologia
9.
Methods Mol Biol ; 2288: 73-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270005

RESUMO

In the context of plant regeneration, in vitro systems to produce embryos are frequently used. In many of these protocols, nonzygotic embryos are initiated that will produce shoot-like structures but may lack a primary root. By increasing the auxin-to-cytokinin ratio in the growth medium, roots are then regenerated in a second step. Therefore, in vitro systems might not or only partially execute a similar developmental program as employed during zygotic embryogenesis. There are, however, in vitro systems that can remarkably mimic zygotic embryogenesis such as Brassica microspore-derived embryos. In this case, the patterning process of these haploid embryos closely follows zygotic embryogenesis and all fundamental tissue types are generated in a rather similar manner. In this review, we discuss the most fundamental molecular events during early zygotic embryogenesis and hope that this brief summary can serve as a reference for studying and developing in vitro embryogenesis systems in the context of doubled haploid production.


Assuntos
Magnoliopsida/embriologia , Padronização Corporal/genética , Padronização Corporal/fisiologia , Brassicaceae/embriologia , Brassicaceae/genética , Brassicaceae/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Ácidos Indolacéticos/metabolismo , Sistema de Sinalização das MAP Quinases , Magnoliopsida/genética , Magnoliopsida/fisiologia , Modelos Biológicos , Biologia Molecular/métodos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Regeneração/genética , Regeneração/fisiologia , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/fisiologia , Zigoto
10.
J Obstet Gynaecol Res ; 47(9): 3232-3240, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34155738

RESUMO

AIM: Abnormal fertilization (1PN/3PN) and its accompanying polar body (PB) conditions have been less discussed in poor ovarian responders. By observing the PBs, we analyzed the mechanisms of abnormal fertilization and aimed to explore the role of intracytoplasmic sperm injection/in vitro fertilization (ICSI/IVF) in POSEIDON group 4 patients. METHODS: An observational study. All fresh IVF/ICSI cycles from January 2018 to December 2019 were evaluated. The inclusion criteria were POSEIDON group 4. Fertilization and PB conditions were assessed 16-18 h post-insemination. Primary observation endpoints including normal fertilization, abnormal fertilization, and total fertilization failure rate. RESULTS: A total of 351 cycles involving 180 patients met the inclusion criteria. Of these, 15 cycles reported no retrieved oocytes. Finally, 336 cycles (IVF, n = 267; ICSI, n = 69) were included. A total of 1005 oocytes and 939 embryos were assessed. The mean female age was 40.8 years, and the mean AMH level was 0.6 ng/mL. The normal fertilization rate was 69.7%. The zygote distribution was 18.7% 0PN, 3.9% 1PN, 66.9% 2PN, 9.5% 3PN, and 1.0% ≥4PN. For 1PN zygotes, 59% were denoted as 1PN2PB. The mean 3PN rate was 8.9%. CONCLUSIONS: In POSEIDON group 4, most of the monopronucleated zygotes were 1PN2PB. Digyny (3PN1PB), due to failure to extrude the second PB, was the major cause of triploidy in which ICSI could not circumvent. The distribution of abnormally fertilized zygotes was similar in IVF and ICSI. To investigate the mechanisms of abnormal fertilization and assess whether ICSI is necessary, analysis of PB will provide important clues.


Assuntos
Corpos Polares , Zigoto , Adulto , Feminino , Fertilização In Vitro , Humanos , Inseminação , Estudos Retrospectivos
12.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070406

RESUMO

The human lifespan is strongly influenced by telomere length (TL) which is defined in a zygote-when two highly specialised haploid cells form a new diploid organism. Although TL is a variable parameter, it fluctuates in a limited range. We aimed to establish the determining factors of TL in chromosomes of maternal and paternal origin in human triploid zygotes. Using Q-FISH, we examined TL in the metaphase chromosomes of 28 human triploid zygotes obtained from 22 couples. The chromosomes' parental origin was identified immunocytochemically through weak DNA methylation and strong hydroxymethylation in the sperm-derived (paternal) chromosomes versus strong DNA methylation and weak hydroxymethylation in the oocyte-derived (maternal) ones. In 24 zygotes, one maternal and two paternal chromosome sets were identified, while the four remaining zygotes contained one paternal and two maternal sets. For each zygote, we compared mean relative TLs between parental chromosomes, identifying a significant difference in favour of the paternal chromosomes, which attests to a certain "imprinting" of these regions. Mean relative TLs in paternal or maternal chromosomes did not correlate with the respective parent's age. Similarly, no correlation was observed between the mean relative TL and sperm quality parameters: concentration, progressive motility and normal morphology. Based on the comparison of TLs in chromosomes inherited from a single individual's gametes with those in chromosomes inherited from different individuals' gametes, we compared intraindividual (intercellular) and interindividual variability, obtaining significance in favour of the latter and thus validating the role of heredity in determining TL in zygotes. A comparison of the interchromatid TL differences across the chromosomes from sets of different parental origin with those from PHA-stimulated lymphocytes showed an absence of a significant difference between the maternal and paternal sets but a significant excess over the lymphocytes. Therefore, interchromatid TL differences are more pronounced in zygotes than in lymphocytes. To summarise, TL in human zygotes is determined both by heredity and parental origin; the input of other factors is possible within the individual's reaction norm.


Assuntos
Cromossomos Humanos/metabolismo , Metáfase , Homeostase do Telômero , Telômero/metabolismo , Triploidia , Zigoto/metabolismo , Fertilização In Vitro , Humanos , Telômero/patologia , Zigoto/patologia
13.
Development ; 148(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34104941

RESUMO

Zygotic genomic activation (ZGA) is a landmark event in the maternal-to-zygotic transition (MZT), and the regulation of ZGA by maternal factors remains to be elucidated. In this study, the depletion of maternal ring finger protein 114 (RNF114), a ubiquitin E3 ligase, led to developmental arrest of two-cell mouse embryos. Using immunofluorescence and transcriptome analysis, RNF114 was proven to play a crucial role in major ZGA. To study the underlying mechanism, we performed protein profiling in mature oocytes and found a potential substrate for RNF114, chromobox 5 (CBX5), ubiquitylation and degradation of which was regulated by RNF114. The overexpression of CBX5 prevented embryonic development and impeded major ZGA. Furthermore, TAB1 was abnormally accumulated in mutant two-cell embryos, which was consistent with the result of in vitro knockdown of Rnf114. Knockdown of Cbx5 or Tab1 in maternal RNF114-depleted embryos partially rescued developmental arrest and the defect of major ZGA. In summary, our study reveals that maternal RNF114 plays a precise role in degrading some important substrates during the MZT, the misregulation of which may impede the appropriate activation of major ZGA in mouse embryos.


Assuntos
Desenvolvimento Embrionário/fisiologia , Genoma , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Zigoto/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Cromossômicas não Histona/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Fatores de Transcrição/metabolismo , Transcriptoma
14.
Development ; 148(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34164654

RESUMO

Understanding the mechanisms of embryonic cell cycles is a central goal of developmental biology, as the regulation of the cell cycle must be closely coordinated with other events during early embryogenesis. Quantitative imaging approaches have recently begun to reveal how the cell cycle oscillator is controlled in space and time, and how it is integrated with mechanical signals to drive morphogenesis. Here, we discuss how the Drosophila embryo has served as an excellent model for addressing the molecular and physical mechanisms of embryonic cell cycles, with comparisons to other model systems to highlight conserved and species-specific mechanisms. We describe how the rapid cleavage divisions characteristic of most metazoan embryos require chemical waves and cytoplasmic flows to coordinate morphogenesis across the large expanse of the embryo. We also outline how, in the late cleavage divisions, the cell cycle is inter-regulated with the activation of gene expression to ensure a reliable maternal-to-zygotic transition. Finally, we discuss how precise transcriptional regulation of the timing of mitosis ensures that tissue morphogenesis and cell proliferation are tightly controlled during gastrulation.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Drosophila/embriologia , Desenvolvimento Embrionário/fisiologia , Animais , Proteína Quinase CDC2 , Ciclo Celular/genética , Proteínas de Drosophila , Embrião de Mamíferos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Mitose , Morfogênese , Xenopus/embriologia , Zigoto/metabolismo
15.
BMC Pregnancy Childbirth ; 21(1): 361, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33952184

RESUMO

BACKGROUND: In assisted reproduction technology embryo competence is routinely evaluated on morphological criteria but efficacy remains relatively low. Additional information could be obtained by evaluating pronuclear (PN) morphology. Up to now controversial results have been reported about the prognostic value of PN score. One of the main limitations of literature data is the use of different PN classification methods. In this regard, in 2011 the ESHRE and Alpha Scientists in Reproductive Medicine defined three PN categories to standardize zygote assessment. In this study we evaluated whether the consensus ESHRE-Alpha system for the pronuclear scoring could be an useful additional criterion to improve prediction of embryo implantation potential. METHODS: This is a retrospective, longitudinal, observational, cohort study. We included 3004 zygotes from 555 women who underwent ICSI treatment at our Center between January 2014 and June 2019. The PN were categorized as score 1: symmetrical, 2: non-symmetrical, 3: abnormal. A subset of 110 zygotes did not cleaved. On day 2-3 1163 embryos were transferred, 232 arrested, and 9 were cryopreserved. Among the 1490 embryos cultured up to day 5-7, 516 became blastocysts: 123 were transferred on day 5 and 393 were cryopreserved. Comparisons of age, cleavage and blastocyst rate, quality of embryos, implantation success among PN score groups were evaluated by chi-square test or Kruskal-Wallis test as appropriate. Potential predictors of embryo implantation were first tested in univariable analysis using generalized estimating equations taking into account correlation between embryos originated from the same patient. Then, variables potentially associated with implantation success (P<0.05) were included in a multivariable analysis for calculating the adjusted odds ratio (OR) and 95% confidence interval (CI). RESULTS: There was no significant difference in patients'age, cleavage and blastulation rates, and embryo morphology among the three PNscore groups. The PN score 1-embryos had a greater implantation success respect to score 2-3-ones (OR 1.83; 95% CI 1.34-2.50, P=0.0001). Consistently, the pronuclear score remained predictive of implantation in top quality embryos (OR 1.68; 95%CI 1.17-2.42, P= 0.005). CONCLUSIONS: The consensus pronuclear score may be routinely included among criteria for embryo evaluation to increase patients' chance of becoming pregnant.


Assuntos
Núcleo Celular/ultraestrutura , Implantação do Embrião , Injeções de Esperma Intracitoplásmicas , Adulto , Análise de Variância , Blastocisto , Fase de Clivagem do Zigoto , Feminino , Humanos , Masculino , Estudos Retrospectivos , Zigoto
16.
Nat Genet ; 53(4): 551-563, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33821005

RESUMO

Polycomb repressive complexes 1 and 2 (PRC1/2) maintain transcriptional silencing of developmental genes largely by catalyzing the formation of mono-ubiquitinated histone H2A at lysine 119 (H2AK119ub1) and trimethylated histone H3 at lysine 27 (H3K27me3), respectively. How Polycomb domains are reprogrammed during mammalian preimplantation development remains largely unclear. Here we show that, although H2AK119ub1 and H3K27me3 are highly colocalized in gametes, they undergo differential reprogramming dynamics following fertilization. H3K27me3 maintains thousands of maternally biased domains until the blastocyst stage, whereas maternally biased H2AK119ub1 distribution in zygotes is largely equalized at the two-cell stage. Notably, while maternal PRC2 depletion has a limited effect on global H2AK119ub1 in early embryos, it disrupts allelic H2AK119ub1 at H3K27me3 imprinting loci including Xist. By contrast, acute H2AK119ub1 depletion in zygotes does not affect H3K27me3 imprinting maintenance, at least by the four-cell stage. Importantly, loss of H2AK119ub1, but not H3K27me3, causes premature activation of developmental genes during zygotic genome activation (ZGA) and subsequent embryonic arrest. Thus, our study reveals distinct dynamics and functions of H3K27me3 and H2AK119ub1 in mouse preimplantation embryos.


Assuntos
Blastocisto/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Herança Materna , RNA Longo não Codificante/genética , Animais , Blastocisto/citologia , Feminino , Fertilização/genética , Histonas/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Herança Paterna , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Gravidez , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Longo não Codificante/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo , Ubiquitinação , Zigoto/citologia , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
17.
Viruses ; 13(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800329

RESUMO

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.


Assuntos
Avulavirus/genética , Avulavirus/isolamento & purificação , Columbidae/virologia , Genoma Viral , Genótipo , Filogenia , Animais , Avulavirus/classificação , Avulavirus/patogenicidade , Galinhas/virologia , Testes de Inibição da Hemaglutinação , Organismos Livres de Patógenos Específicos , Vitória , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Virulência , Zigoto/virologia
18.
Dev Biol ; 476: 249-258, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33905721

RESUMO

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.


Assuntos
Coturnix/embriologia , Coturnix/genética , Ciclina D1/genética , Animais , Blastoderma/embriologia , Blastoderma/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Ciclina D1/metabolismo , Desenvolvimento Embrionário/genética , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , RNA Mensageiro/genética , Ativação Transcricional/genética , Zigoto/metabolismo
19.
J Immunol ; 206(9): 2001-2014, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33858963

RESUMO

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.


Assuntos
Resistência à Doença/imunologia , Doenças dos Peixes/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Isotipos de Imunoglobulinas/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/fisiologia , Animais , Resistência à Doença/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/imunologia , Embrião não Mamífero/microbiologia , Feminino , Doenças dos Peixes/microbiologia , Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Isotipos de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/metabolismo , Masculino , Herança Materna/genética , Herança Materna/imunologia , Vibrio/classificação , Vibrio/imunologia , Vibrio/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/imunologia , Zigoto/metabolismo , Zigoto/microbiologia
20.
Adv Exp Med Biol ; 1310: 551-564, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33834450

RESUMO

Genetically engineered mouse (GEM) models have been revolutionizing the biomedical studies on deciphering the physiological roles of genes in vivo. In addition to deactivating a gene in mice, diverse strategies have been created to monitor gene expressions and molecular dynamics of specific proteins in vivo. Although gene targeting in mouse embryonic stem (ES) cells was essential for the precise engineering of the mouse genome over almost three decades, this process is a time-consuming, expensive, and laborious one. These days, new technologies that directly apply engineered endonucleases, such as zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector (TALE) Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, into the mouse zygotes are enabling us to rapidly replace conventional gene targeting in mouse ES cells. In this chapter, we will describe the principles of reporter mouse strains and the recent advances in generating them using engineered endonucleases.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endonucleases/genética , Edição de Genes , Marcação de Genes , Engenharia Genética , Camundongos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Zigoto
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