RESUMO
Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.
Assuntos
Actinas , Núcleo Celular , Actinas/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Ciclo CelularRESUMO
Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.
Assuntos
Núcleo Celular/metabolismo , Reprogramação Celular , Cromatina/metabolismo , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Técnicas de Transferência Nuclear , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Linhagem Celular , Cromatina/genética , Montagem e Desmontagem da Cromatina , Clonagem Molecular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Oócitos , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Xenopus laevisRESUMO
Differentiated cells can be experimentally reprogrammed back to pluripotency by nuclear transfer, cell fusion or induced pluripotent stem cell technology. Nuclear transfer and cell fusion can lead to efficient reprogramming of gene expression. The egg and oocyte reprogramming process includes the exchange of somatic proteins for oocyte proteins, the post-translational modification of histones and the demethylation of DNA. These events occur in an ordered manner and on a defined timescale, indicating that reprogramming by nuclear transfer and by cell fusion rely on deterministic processes.
Assuntos
Núcleo Celular/metabolismo , Reprogramação Celular , Oócitos/metabolismo , Óvulo/metabolismo , Animais , Desdiferenciação Celular , Fusão Celular , Núcleo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Feminino , Expressão Gênica , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Metilação , Técnicas de Transferência Nuclear , Oócitos/citologia , Óvulo/citologia , Fatores de Tempo , Xenopus laevisRESUMO
Nuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner. We demonstrate that an extensive exchange of somatic- for oocyte-specific factors mediates reprogramming and leads to robust oocyte RNA polymerase II binding and phosphorylation on transplanted chromatin. Moreover, genome-wide binding of oocyte-specific linker histone B4 supports its role in transcriptional reprogramming. Thus, our study reveals the rapid, abundant, and stepwise loading of oocyte-specific factors onto somatic chromatin as important determinants for successful reprogramming.
Assuntos
Reprogramação Celular/genética , Cromatina/metabolismo , Histonas/fisiologia , Oócitos/metabolismo , Xenopus/embriologia , Animais , Células Cultivadas , Reprogramação Celular/fisiologia , Genoma , Camundongos , Técnicas de Transferência Nuclear , Especificidade de Órgãos , RNA/genética , Análise de Sequência de RNA , Xenopus/genéticaRESUMO
Purpose: In vitro maturation (IVM) of human oocytes offers an invaluable opportunity for infertility treatment. However, in vitro matured oocytes often show lower developmental abilities than their in vivo counterparts, and molecular mechanisms underlying successful maturation remain unclear. In this study, we investigated gene expression profiles of in vitro matured oocytes at the single-cell level to gain mechanistic insight into IVM of human oocytes. Methods: Human oocytes were retrieved by follicular puncture and in vitro matured. In total, 19 oocytes from 11 patients were collected and subjected to single-cell RNA-seq analyses. Results: Global gene expression profiles were similar among oocytes at the same maturation stage, while a small number of oocytes showed distinct transcriptomes from those at the corresponding maturation stage. Differential gene expression analysis identified hundreds of transcripts that dynamically altered their expression during IVM, and we revealed molecular pathways and upstream regulators that may govern oocyte maturation. Furthermore, oocytes that were delayed in their maturation showed distinct transcriptomes. Finally, we identified genes whose transcripts were enriched in each stage of oocyte maturation. Conclusions: Our work uncovers transcriptomic changes during human oocyte IVM and the differential gene expression profile of each oocyte.
RESUMO
In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.
Assuntos
Inibidores de Histona Desacetilases/química , Técnicas de Transferência Nuclear/instrumentação , Oócitos/química , Animais , Inibidores de Histona Desacetilases/classificação , Camundongos , Peptídeos Cíclicos/químicaRESUMO
BACKGROUND: Ectopic gas in the graft is occasionally encountered upon follow-up computed tomography (CT) after anterior cervical corpectomy and fusion (ACCF). However, most cases lack inflammatory responses and manifestations of infection. Although the clinical significance of ectopic gas in the graft has not yet been established, to the best of our knowledge, no previous studies have described ectopic gas in the graft after ACCF. This study evaluated ectopic gas in the fibular graft upon follow-up CT after ACCF. METHODS: We reviewed 112 patients who underwent ACCF and follow-up CT, with a minimum follow-up period of 3 years. CT images were retrospectively reviewed to confirm the presence of ectopic gas in the graft and bone fusion. Bone fusion was defined as follows: mobility less than 2 mm between spinous processes on the flection-extension radiograph or a bone bridge on CT images. RESULTS: Of the 112 patients, 30 (27%) patients had ectopic gas in the fibular grafts. Among them, ectopic gas was initially observed 3 months after surgery (early onset) in 23 (77%) patients and 6 months after surgery (late-onset) in the remaining seven (23%) patients. Upon the latest follow-up CT, ectopic gas more frequently remained in late-onset (4/7, 57%) rather than in early-onset (3/23, 13%) cases (p = 0.033). Bone fusion was not observed when CT images exhibited ectopic gas in the graft, whereas ectopic gas was not observed when CT images exhibited bone fusion. CONCLUSION: Ectopic gas in the fibular graft was observed at both early and late-onset after ACCF; late-onset gas remained significantly. The remaining gas was strongly associated with pseudoarthrosis; therefore, pseudoarthrosis should be considered when ectopic gas in the graft is observed on CT images.
Assuntos
Vértebras Cervicais , Fusão Vertebral , Transplante Ósseo , Vértebras Cervicais/diagnóstico por imagem , Vértebras Cervicais/cirurgia , Fíbula/diagnóstico por imagem , Fíbula/cirurgia , Humanos , Estudos Retrospectivos , Fusão Vertebral/efeitos adversos , Resultado do TratamentoRESUMO
For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.
Assuntos
Metilação de DNA/genética , Epigênese Genética , Histona-Lisina N-Metiltransferase/genética , Espermatozoides/metabolismo , Animais , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/biossíntese , Histonas , Humanos , Masculino , Ranidae/genética , Ranidae/crescimento & desenvolvimento , Espermátides/crescimento & desenvolvimento , Espermátides/metabolismo , Espermatozoides/crescimento & desenvolvimentoRESUMO
Maternal factors stored in eggs and oocytes are necessary for reprogramming sperm for embryonic development. This reprogramming activity of maternal factors also works towards somatic cells, including terminally differentiated cells. Several different experimental systems utilizing egg and oocyte materials have been applied to study nuclear reprogramming by maternal factors. Among these systems, the most widely used is the transfer of a somatic cell nucleus to an oocyte arrested at the metaphase II stage, leading to the production of a cloned animal. Nuclear transfer to an unfertilized oocyte thus provides a unique opportunity to examine reprogramming processes involved in acquiring totipotency. Other experimental systems are also available to study maternal reprogramming, such as nuclear transfer to Xenopus laevis oocytes at the germinal vesicle stage, treatment with extracts obtained from eggs or oocytes, and induced pluripotency with overexpressed maternal factors. Each system can be used for answering different types of scientific questions. This review describes currently available reprogramming systems using egg and oocyte materials and discusses how we can deepen our understanding of reprogramming mechanisms by taking advantage of these various experimental systems.
Assuntos
Reprogramação Celular , Metáfase , Técnicas de Transferência Nuclear , Oócitos/citologia , Animais , Núcleo Celular/metabolismo , Feminino , Histonas/metabolismo , Humanos , Xenopus , Xenopus laevisRESUMO
Amphibian oocytes can rapidly and efficiently reprogram the transcription of transplanted somatic nuclei. To explore the factors and mechanisms involved, we focused on nuclear actin, an especially abundant component of the oocyte's nucleus (the germinal vesicle). The existence and significance of nuclear actin has long been debated. Here, we found that nuclear actin polymerization plays an essential part in the transcriptional reactivation of the pluripotency gene Oct4 (also known as Pou5f1). We also found that an actin signaling protein, Toca-1, enhances Oct4 reactivation by regulating nuclear actin polymerization. Toca-1 overexpression has an effect on the chromatin state of transplanted nuclei, including the enhanced binding of nuclear actin to gene regulatory regions. This is the first report showing that naturally stored actin in an oocyte nucleus helps transcriptional reprogramming in a polymerization-dependent manner.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Reprogramação Celular , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Oócitos/metabolismo , Xenopus , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a Ácido Graxo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Polimerização , Transdução de Sinais , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Maternal RNA/protein degradation and zygotic genome activation (ZGA), occurring during maternal-to-zygotic transition (MZT), are the first essential events for the development of pre-implantation embryos. Previously, we have shown the importance of the ubiquitin-proteasome system (UPS) for initiation of minor ZGA at the 1-cell stage of mouse embryos. However, little is known about the mechanism of involvement of the UPS-degraded maternal proteins in ZGA. In this study, we investigated the effect of inhibiting maternal protein degradation by the reversible proteasome inhibitor, MG132, on post-implantation development and ZGA regulation during early cleavage stages. Our study revealed that zygotic transcription by RNA polymerase II (Pol II) at the 1-cell stage was delayed and the full-term development was affected by transient proteasome inhibition during 1 to 9 h post-insemination (hpi). Furthermore, we found that the transient inhibition of proteasome activity at the 2-cell stage delayed the onset of transcription of some major ZGA genes. These results support the model hypothesizing the requirement of sequential degradation of maternal proteins by UPS for the proper onset of ZGA and normal progression of MZT in early mouse embryos.
Assuntos
Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina/genética , Animais , Camundongos , Oócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismoRESUMO
Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.
Assuntos
Núcleo Celular/enzimologia , Metilação de DNA , Ectogênese , Epigênese Genética , Peroxirredoxinas/metabolismo , Zigoto/enzimologia , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Metilação de DNA/efeitos dos fármacos , Ectogênese/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Feminino , Fertilização in vitro , Peróxido de Hidrogênio/toxicidade , Masculino , Camundongos Endogâmicos ICR , Microscopia Confocal , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Zigoto/citologia , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimentoRESUMO
Although populations of the coconut crab, Birgus latro, have declined in the tropical Indo-Pacific region, insufficient knowledge exists for the management of this species. We investigated the growth of the northernmost coconut crab population, located at Ocean Expo Park, Okinawa, southern Japan, using a mark-recapture method based on the identification of individual carapace grooving patterns. Of the 485 crabs photographed (264 males, 221 females; 14.3-68.8 mm thoracic length [ThL]), 64 males and 62 females were recaptured (recapture rate 26%). The liberty period ranged from two to 2384 days. The annual data indicated that most crabs molted during winter, except for juveniles and crabs near the maximum size. Using the GROTAG program, the asymptotic ThL (L∞) was estimated as 80.72 and 49.89 mm for males and females, respectively. The Brody growth coefficient (K) was 0.063 for both sexes. The growth curves from these parameters showed that males grew larger than females because of a difference in growth speed. Longevity was estimated at approximately 50 years for both sexes. The growth data obtained in the present study, which are the most precise gathered for the coconut crab to date, can be compared with the results of studies performed in other regions.
Assuntos
Distribuição Animal/fisiologia , Sistemas de Identificação Animal , Braquiúros/anatomia & histologia , Braquiúros/crescimento & desenvolvimento , Animais , Braquiúros/fisiologia , Feminino , MasculinoRESUMO
STUDY DESIGN: A retrospective study of 58 patients undergoing cantilever transforaminal lumbar interbody fusion (c-TLIF). OBJECTIVES: To evaluate morphologic changes in the intervertebral foramen (IVF) on the side contralateral to spacer insertion in patients undergoing c-TLIF using plain x-ray films and computed tomography scan. SUMMARY OF BACKGROUND DATA: The morphologic changes in the contralateral lumbar foramen in c-TLIF using unilateral insertion of spacers have not been well studied. MATERIALS AND METHODS: Fifty-eight consecutive patients with lumbar dysplastic changes or degenerative disk diseases underwent c-TLIF using 96 kidney-type spacers with local bone grafts. Radiographic findings (sagittal disk angle), computed tomography scan findings (coronal disk angle, disk height, foraminal height (FH), foraminal width, and cross-sectional area of IVF in contralateral lumbar foramen) were compared between preoperative period and 6 months after surgery. The correlations between contralateral lumbar foraminal dimensions and disk height, sagittal disk angle, and coronal disk angle were analyzed. RESULTS: After c-TLIF, sagittal angle, disk height, FH, foraminal width, and cross-sectional area of the IVF were significantly increased. Increase in posterior disk height showed a positive correlation with increases in FH, foraminal width, and cross-sectional area of IVF (r=0.235-0.511). However, the increase in sagittal disk angle showed a negative correlation with changes in foraminal width and cross-sectional area of IVF (r=-0.256 to -0.206). CONCLUSIONS: Lumbar foraminal dimensions on the side contralateral to spacer insertion increased significantly after c-TLIF, suggesting that c-TLIF enables indirect decompression of the contralateral nerve root. Although increase in posterior disk height was shown to be an important factor to increase contralateral foraminal size, segmental lordosis was a risk factor for a decrease in contralateral foraminal size.
Assuntos
Fixadores Internos , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/cirurgia , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Fusão Vertebral/instrumentação , Adulto , Idoso , Anatomia Transversal , Feminino , Humanos , Disco Intervertebral/diagnóstico por imagem , Lordose/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Raízes Nervosas Espinhais/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
Patient-specific somatic cell reprogramming is likely to have a large impact on medicine by providing a source of cells for disease modelling and regenerative medicine. Several strategies can be used to reprogram cells, yet they are generally characterised by a low reprogramming efficiency, reflecting the remarkable stability of the differentiated state. Transcription factors, chromatin modifications, and noncoding RNAs can increase the efficiency of reprogramming. However, the success of nuclear reprogramming is limited by epigenetic mechanisms that stabilise the state of gene expression in somatic cells and thereby resist efficient reprogramming. We review here the factors that influence reprogramming efficiency, especially those that restrict the natural reprogramming mechanisms of eggs and oocytes. We see this as a step towards understanding the mechanisms by which nuclear reprogramming takes place.
Assuntos
Reprogramação Celular , Epigênese Genética , Animais , Divisão Celular , Metilação de DNA , Humanos , Modelos Genéticos , Transcrição GênicaRESUMO
Proper regulation of transcription is essential for cells to acquire and maintain cell identity. Transcriptional activation plays a central role in gene regulation and can be modulated by introducing transcriptional activators such as transcription factors. Activators act on their specific target genes to induce transcription. Reprogramming experiments have revealed that as cells become differentiated, some genes are highly silenced and even introduction of activators that target these silenced genes does not induce transcription. This can be explained by chromatin-based repression that restricts access of transcriptional activators to silenced genes. Transcriptional activation from these genes can be accomplished by opening chromatin, in addition to providing activators. Once a de novo transcription network is established, cells are differentiated or reprogrammed to a new cell type. Emerging evidence suggests that actin in the nucleus (nuclear actin) and nuclear actin-binding proteins are implicated in these transcriptional regulatory processes. This review summarizes roles of nuclear actin and actin-binding proteins in transcriptional regulation. We also discuss possible functions of nuclear actin during reprogramming in the context of transcription and chromatin remodeling.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Reprogramação Celular , Regulação da Expressão Gênica , Proteínas dos Microfilamentos/metabolismo , Transcrição Gênica , Animais , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Drosophila melanogaster , Inativação Gênica , Humanos , Camundongos , Oócitos/citologia , Ativação Transcricional , Xenopus/metabolismoRESUMO
Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.
Assuntos
Blastocisto/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Técnicas de Transferência Nuclear , Oócitos/química , Oócitos/metabolismo , Animais , Blastocisto/citologia , Feminino , Metáfase/fisiologia , Oócitos/citologia , Suínos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.
Assuntos
Cromatina/metabolismo , Proteínas do Ovo/metabolismo , Proteínas Nucleares/metabolismo , Interações Espermatozoide-Óvulo , Espermátides/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Eletroforese em Gel de Poliacrilamida , Feminino , Immunoblotting , Masculino , Espectrometria de Massas , Proteínas Nucleares/isolamento & purificação , Ligação Proteica , Mapeamento de Interação de Proteínas , Isoformas de Proteínas , Extratos de Tecidos , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis/metabolismoRESUMO
Decidualization of the human endometrium is critical for establishing pregnancy and is entailed by differentiation of endometrial stromal cells (ESCs) into decidual cells. During decidualization, the actin cytoskeleton is dynamically reorganized for the ESCs' morphological and functional changes. Although actin dynamically alters its polymerized state upon external stimuli not only in the cytoplasm, but also in the nucleus, nuclear actin dynamics during decidualization have not been elucidated. Here, we show that nuclear actin was specifically assembled during decidualization of human ESCs. This decidualization-specific formation of nuclear actin filaments was disassembled following the withdrawal of the decidualization stimulus, suggesting its reversible process. Mechanistically, RNA-seq analyses revealed that the forced disassembly of nuclear actin resulted in the suppression of decidualization, accompanied with the abnormal upregulation of cell proliferation genes, leading to incomplete cell cycle arrest. CCAAT/enhancer-binding protein beta (C/EBPß), an important regulator for decidualization, was responsible for downregulation of the nuclear actin exporter, thus accelerating nuclear actin accumulation and its assembly for decidualization. Taken together, we demonstrate that decidualization-specific nuclear actin assembly induces cell cycle arrest for establishing the decidualized state of ESCs. We propose that not only the cytoplasmic actin, but also nuclear actin dynamics profoundly affect decidualization process in humans for ensuring pregnancy.
Assuntos
Actinas , Núcleo Celular , Decídua , Endométrio , Células Estromais , Humanos , Feminino , Células Estromais/metabolismo , Actinas/metabolismo , Endométrio/citologia , Endométrio/metabolismo , Decídua/metabolismo , Decídua/citologia , Núcleo Celular/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Gravidez , Diferenciação Celular , Proliferação de Células , Citoesqueleto de Actina/metabolismoRESUMO
PURPOSE: Alterations of three-dimensional cervical curvature in conventional anterior cervical approach position are not well understood. The purpose of this study was to evaluate alignment changes of the cervical spine in the position. In addition, simulated corpectomy was evaluated with regard to sufficiency of decompression and perforation of the vertebral artery canal. METHODS: Fifty patients with cervical spinal disorders participated. Cervical CT scanning was performed in the neutral and supine position (N-position) and in extension and right rotation simulating the conventional anterior approach position (ER-position). Rotation at each vertebral level was measured. With simulation of anterior corpectomy in a vertical direction with a width of 17 mm, decompression width at the posterior wall of the vertebrae and the distance from each foramen of the vertebral artery (VA) were measured. RESULTS: In the ER-position, the cervical spine was rotated rightward by 37.2° ± 6.2° between the occipital bone and C7. While the cervical spine was mainly rotated at C1/2, the subaxial vertebrae were also rotated by several degrees. Due to the subaxial rotation, the simulated corpectomy resulted in smaller decompression width on the left side and came closer to the VA canal on the right side. CONCLUSIONS: In the ER-position, the degrees of right rotation of subaxial vertebrae were small but significant. Therefore, preoperative understanding of this alteration of cervical alignment is essential for performing safe and sufficient anterior corpectomy of the cervical spine.