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1.
Cell Prolif ; 52(5): e12657, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31264311

RESUMO

OBJECTIVES: A high rate of chromosome aneuploidy is exhibited in in vitro fertilization (IVF)-derived embryos. Our previous experiments suggested that reactive oxygen species (ROS) can activate Mad2, a key protein in the spindle assembly checkpoint (SAC), and delay the first mitotic, providing time to prevent the formation of embryonic aneuploidy. We aimed to determine whether mitotic kinase Aurora B was involved in the SAC function to prevent aneuploidy in IVF-derived embryos. MATERIALS AND METHODS: We analysed aneuploidy formation and repair during embryo pre-implantation via 4',6-diamidino-2-phenylindole (DAPI) staining and karyotype analysis. We assessed Aurora B activation by immunofluorescence and investigated the effect of Aurora B inhibition on embryo injury-related variables, such as embryonic development, ROS levels, mitochondrial membrane potential and γH2AX-positive expression. RESULTS: We observed the expression and phosphorylation of Thr232 in Aurora B in oxidative stress-induced zygotes. Moreover, inhibition of Aurora B caused chromosome mis-segregation, abnormal spindle structures, abnormal chromosome number and reduced expression of Mad2 in IVF embryos. Our results suggest that Aurora B causes mitotic arrest and participates in SAC via Mad2 and H3S10P, which is required for self-correction of aneuploidies. CONCLUSIONS: We demonstrate here that oxidative stress-induced DNA damage triggers Aurora B-mediated activation of SAC, which prevents aneuploidy at the first mitotic cleavage in early mouse IVF embryos.


Assuntos
Aurora Quinase B/metabolismo , Proteínas Mad2/metabolismo , Aneuploidia , Animais , Aurora Quinase B/antagonistas & inibidores , Segregação de Cromossomos/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Peróxido de Hidrogênio/farmacologia , Pontos de Checagem da Fase M do Ciclo Celular , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitose , Organofosfatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Quinazolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Zigoto/metabolismo
2.
Nat Commun ; 10(1): 2792, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243271

RESUMO

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.


Assuntos
Embrião de Mamíferos/metabolismo , Análise de Sequência de RNA , Transcrição Genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Mutação , Transcriptoma
3.
BMC Genomics ; 20(1): 439, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151386

RESUMO

BACKGROUND: The last decade witnessed a number of genome-wide studies on human pre-implantation, which mostly focused on genes and provided only limited information on repeats, excluding the satellites. Considering the fact that repeats constitute a large portion of our genome with reported links to human physiology and disease, a thorough understanding of their spatiotemporal regulation during human embryogenesis will give invaluable clues on chromatin dynamics across time and space. Therefore, we performed a detailed expression analysis of all repetitive DNA elements including the satellites across stages of human pre-implantation and embryonic stem cells. RESULTS: We uncovered stage-specific expressions of more than a thousand repeat elements whose expressions fluctuated with a mild global decrease at the blastocyst stage. Most satellites were highly expressed at the 4-cell level and expressions of ACRO1 and D20S16 specifically peaked at this point. Whereas all members of the SVA elements were highly upregulated at 8-cell and morula stages, other transposons and small RNA repeats exhibited a high level of variation among their specific subtypes. Our repeat enrichment analysis in gene promoters coupled with expression correlations highlighted potential links between repeat expressions and nearby genes, emphasising mostly 8-cell and morula specific genes together with SVA_D, LTR5_Hs and LTR70 transposons. The DNA methylation analysis further complemented the understanding on the mechanistic aspects of the repeatome's regulation per se and revealed critical stages where DNA methylation levels are negatively correlating with repeat expression. CONCLUSIONS: Taken together, our study shows that specific expression patterns are not exclusive to genes and long non-coding RNAs but the repeatome also exhibits an intriguingly dynamic pattern at the global scale. Repeats identified in this study; particularly satellites, which were historically associated with heterochromatin, and those with potential links to nearby gene expression provide valuable insights into the understanding of key events in genomic regulation and warrant further research in epigenetics, genomics and developmental biology.


Assuntos
DNA/química , Desenvolvimento Embrionário/genética , Expressão Gênica , Sequências Repetitivas de Ácido Nucleico , Metilação de DNA , DNA Satélite/química , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Humanos , Elementos Nucleotídeos Longos e Dispersos , Elementos Nucleotídeos Curtos e Dispersos
4.
Nat Commun ; 10(1): 2487, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171776

RESUMO

Lack or excess expression of the surface ectoderm-expressed transcription factor Grainyhead-like2 (Grhl2), each prevent spinal neural tube closure. Here we investigate the causative mechanisms and find reciprocal dysregulation of epithelial genes, cell junction components and actomyosin properties in Grhl2 null and over-expressing embryos. Grhl2 null surface ectoderm shows a shift from epithelial to neuroepithelial identity (with ectopic expression of N-cadherin and Sox2), actomyosin disorganisation, cell shape changes and diminished resistance to neural fold recoil upon ablation of the closure point. In contrast, excessive abundance of Grhl2 generates a super-epithelial surface ectoderm, in which up-regulation of cell-cell junction proteins is associated with an actomyosin-dependent increase in local mechanical stress. This is compatible with apposition of the neural folds but not with progression of closure, unless myosin activity is inhibited. Overall, our findings suggest that Grhl2 plays a crucial role in regulating biomechanical properties of the surface ectoderm that are essential for spinal neurulation.


Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Tubo Neural/embriologia , Células Neuroepiteliais/metabolismo , Neurulação/genética , Fatores de Transcrição/genética , Actomiosina/genética , Actomiosina/metabolismo , Animais , Fenômenos Biomecânicos , Caderinas/metabolismo , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Células Epiteliais/metabolismo , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Camundongos , Tubo Neural/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Estresse Mecânico , Fatores de Transcrição/metabolismo
5.
Nature ; 570(7759): 77-82, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086336

RESUMO

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fertilização , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
6.
Horm Metab Res ; 51(5): 315-325, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31071736

RESUMO

BACKGROUND: The objective of the work was to investigate the cycle characteristics and outcomes of infertile women with nonclassic 21-hydroxylase deficiency (21-OHD) undergoing in vitro fertilization (IVF). METHODS: Twenty-five infertile nonclassic 21-OHD patients were retrospectively observed. From cycle day 3, patients were given human menopausal gonadotropin (HMG) 150 IU/d or 225 IU/d daily. Dexamethasone was administered orally at 0.75 mg/d. Ovulation was co-triggered by human chorionic gonadotropin (hCG) and triptorelin. Binary logistic regression was performed to quantify the effect of IVF parameters on embryo transfer cycle outcomes. The receiver operating characteristic (ROC) curve was used to determine cutoff points of the selected confounders for predicting pregnant probabilities. RESULTS: In controlled ovarian hyperstimulation (COH) cycles, there was a trend that the viable embryos group consisted of more polycystic ovary (PCO) patients. In embryo transfer (ET) cycles, differences were detected in the usage rate of dexamethasone and the minimum progesterone (P4) and total testosterone (TT) values between the non-pregnant group and the biochemical pregnant group. Binary logistic regression analysis confirmed that decreasing minimum P4 and body mass index (BMI) value was respectively correlated with increasing pregnancy probability. ROC analysis proved that the cutoff values for minimum P4 and BMI were 0.45 ng/ml and 23.36 kg/m2. CONCLUSION: In COH cycles, the ultrasonographic appearance of ovary helps to predict the number of viable embryos. In ET cycles, dexamethasone obviously improves the pregnancy rate. If the minimum P4 value before endometrial transformation cannot be kept below 0.45 ng/ml, we may consider cycle cancellation. Moreover, it is suggested that BMI of nonclassic 21-OHD women is regulated below 23.36 kg/m2.


Assuntos
Hiperplasia Suprarrenal Congênita/patologia , Fertilização In Vitro , Indução da Ovulação , Resultado da Gravidez , Adulto , Índice de Massa Corporal , Transferência Embrionária , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Gravidez , Curva ROC
7.
BMC Genomics ; 20(1): 386, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101013

RESUMO

BACKGROUND: Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development. RESULTS: We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development. We have measured differences in gene expression between control and embryos injected with Gam1 mRNA at three developmental stages: early gastrula (immediately following the initiation of zygotic transcription), late gastrula (completion of the formation of the three primary germ layers), and early neurula (appearance of the neural plate). Although changes in gene expression are widespread and can be linked to many biological processes, three pathways, non-canonical Wnt/PCP, snail/twist, and Ets-1, are especially sensitive to the loss of SUMOylation activity and can largely account for the predominant phenotypes of Gam1 embryos. SUMOylation appears to generate different pools of a given transcription factor having different specificities with this post-translational modification involved in the regulation of more complex, as opposed to housekeeping, processes. CONCLUSIONS: We have identified changes in gene expression that underlie the neural tube and heart phenotypes resulting from depressed SUMOylation activity. Notably, these developmental defects correspond to the two most frequently occurring congenital birth defects in humans, strongly suggesting that perturbation of SUMOylation, either globally or of a specific protein, may frequently be the origin of these pathologies.


Assuntos
Embrião de Mamíferos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Defeitos do Tubo Neural/genética , Sumoilação , Proteínas de Xenopus/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica , Cardiopatias Congênitas/patologia , Masculino , Defeitos do Tubo Neural/patologia , Proteínas Virais/administração & dosagem , Xenopus laevis
8.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035421

RESUMO

Embryo implantation in the mink follows the pattern of many carnivores, in that preimplantation embryo diapause occurs in every gestation. Details of the gene expression and regulatory networks that terminate embryo diapause remain poorly understood. Illumina RNA-Seq was used to analyze global gene expression changes in the mink uterus during embryo diapause and activation leading to implantation. More than 50 million high quality reads were generated, and assembled into 170,984 unigenes. A total of 1684 differential expressed genes (DEGs) in uteri with blastocysts in diapause were compared to the activated embryo group (p < 0.05). Among these transcripts, 1527 were annotated as known genes, including 963 up-regulated and 564 down-regulated genes. The gene ontology terms for the observed DEGs, included cellular communication, phosphatase activity, extracellular matrix and G-protein couple receptor activity. The KEGG pathways, including PI3K-Akt signaling pathway, focal adhesion and extracellular matrix (ECM)-receptor interactions were the most enriched. A protein-protein interaction (PPI) network was constructed, and hub nodes such as VEGFA, EGF, AKT, IGF1, PIK3C and CCND1 with high degrees of connectivity represent gene clusters expected to play an important role in embryo activation. These results provide novel information for understanding the molecular mechanisms of maternal regulation of embryo activation in mink.


Assuntos
Blastocisto/metabolismo , Útero/metabolismo , Animais , Blastocisto/fisiologia , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Vison , Gravidez , Transcriptoma/genética , Útero/fisiologia
9.
Methods Mol Biol ; 1965: 261-279, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069681

RESUMO

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin). The methods are simple and can provide complimentary functional and structural information in studies of the effects of embryotoxicants on visceral yolk sac function.


Assuntos
Embrião de Mamíferos/citologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes/metabolismo , Albumina Sérica/metabolismo , Saco Vitelino/metabolismo , Animais , Técnicas de Cultura Embrionária , Embrião de Mamíferos/metabolismo , Endocitose , Fluoresceína-5-Isotiocianato/metabolismo , Microscopia de Fluorescência , Proteólise , Ratos
10.
Methods Mol Biol ; 1965: 297-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069683

RESUMO

BACKGROUND: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde-fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, both of which correlate to apoptotic activity. Thus, LT is a good indicator of apoptosis visualized by confocal microscopy. Results of LT staining of apoptotic cell death correlate well with other whole mount apoptosis vital dyes such as Nile blue sulfate and neutral red, with the added benefit of being fixable in situ. Nile blue sulfate can also be used as a non-vital, nonspecific dye to visualize general morphology. Stains such as acridine orange can be used for surface staining of fixed embryos to yield confocal images that are similar to scanning electron micrographs. METHODS: Mouse embryos were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Embryos are mounted on depression slides, and serial sections are imaged by confocal microscopy, followed by 3-D reconstruction. RESULTS: Embryos or tissues as thick as 500 microns (µm) can be visualized after clearing with BABB. LysoTracker staining reveals apoptotic regions in organogenesis-stage mouse embryos. Morphological observation of tissue was facilitated by combining autofluorescence with Nile blue sulfate staining of fixed embryos or opaque surface staining with acridine orange staining. CONCLUSIONS: The use of BABB for clearing LT vital-stained and fixed embryos matches the refractive index of the tissue to the suspending medium, allowing increased penetration of laser light in a confocal microscope. Nile blue sulfate used as a non-vital dye provides a nonspecific staining of fixed embryos that can then be cleared with methyl salicylate for confocal observation. Sample preparation and staining procedures described here, with optimization of confocal laser scanning microscopy, allow for the detection and visualization of morphological structure and apoptosis in embryos up to 500 µm thick, and stained specimens can be fixed and mounted on depression slides.


Assuntos
Embrião de Mamíferos/ultraestrutura , Lisossomos/metabolismo , Organogênese , Aminas/metabolismo , Animais , Apoptose , Embrião de Mamíferos/metabolismo , Imagem Tridimensional , Camundongos , Microscopia Confocal , Fagocitose
11.
Methods Mol Biol ; 1965: 375-388, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069687

RESUMO

The electrophoretic mobility shift assay (EMSA) is a sensitive and relatively straightforward methodology used to detect sequence-specific DNA-protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein-nucleic acid complexes. Classically, nuclear proteins and DNA are combined, and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the 32P-labelled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the "super-shift", is also presented. The important points are underscored, and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.


Assuntos
DNA/metabolismo , Embrião de Mamíferos/citologia , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismo , Animais , DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Radioisótopos de Fósforo/química , Ligação Proteica , Ratos
12.
Yonsei Med J ; 60(5): 461-466, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31016908

RESUMO

PURPOSE: To investigate the associations between sperm DNA fragmentation (SDF) and embryo formation rate in normal responder women to in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). MATERIALS AND METHODS: Fifty-three consecutive, fresh IVF/ICSI cycles performed from 2014 to 2017 were selected. All women were normal responders (4 to 14 mature oocytes were retrieved) and at least one normally fertilized oocyte with two pronuclei was obtained in all cycles. Semen was collected on the day of oocyte retrieval, and SDF levels were measured by sperm chromatin dispersion test (Halosperm assay). At day 3 after insemination, embryo quality was evaluated by morphologic criteria and categorized as A/B/C/D. Top quality embryo were defined as grade A embryos with seven cells or more. RESULTS: SDF levels showed a positive linear correlation with the male's age (r=0.307, p=0.025) and a negative linear correlation with sperm motility (r=-0.491, p<0.0001). To achieve top-quality or a grade A embryo formation rate >70%, the cut-off value SDF was <30.7% for each. Among individuals with SDF <30.7%, the median top-quality or grade A embryo formation rate was significantly higher than that among individuals with SDF ≥30.7% (38.1% vs. 20.0%, p=0.038; 50% vs. 25.0%, p=0.017). CONCLUSION: In normal responder women, high SDF level resulted in low day 3 embryo formation rates. Our results suggest a paternal effect on embryo quality in IVF/ICSI cycles.


Assuntos
Fragmentação do DNA , Embrião de Mamíferos/metabolismo , Fertilização In Vitro , Injeções de Esperma Intracitoplásmicas , Espermatozoides/metabolismo , Adulto , Feminino , Humanos , Masculino
13.
Dev Cell ; 48(6): 751-763, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30913407

RESUMO

Research in developmental biology has been recently enriched by a multitude of in vitro models recapitulating key milestones of mammalian embryogenesis. These models obviate the challenge posed by the inaccessibility of implanted embryos, multiply experimental opportunities, and favor approaches traditionally associated with organoids and tissue engineering. Here, we provide a perspective on how these models can be applied to study the mechano-geometrical contributions to early mammalian development, which still escape direct verification in species that develop in utero. We thus outline new avenues for robust and scalable perturbation of geometry and mechanics in ways traditionally limited to non-implanting developmental models.


Assuntos
Bioengenharia , Biofísica , Desenvolvimento Embrionário , Mamíferos/embriologia , Modelos Biológicos , Animais , Embrião de Mamíferos/metabolismo , Humanos
14.
Mol Med Rep ; 19(5): 4401-4406, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896847

RESUMO

The objective of the present study was to investigate the effects of for chlorfenuron (FCF) interference with the septin protein on early stage embryos in mice. The 1­cell embryos were collected and divided into an FCF interference group and a control group. The FCF interference group was cultured in FCF media and the control group was cultured in dimethyl sulphoxide media at 37˚C with 5% CO2 until the desired phase was achieved. Septin2 protein expression was detected using immunofluorescence and western blot analysis. Blastocyst α­tubulin was stained by immunofluorescence to observe the alterations in spindles and microtubules. The rate of early embryo development into blastocysts was significantly reduced following FCF treatment (P<0.05). In the control group, septin2 was observed with a confocal microscope; septin2 was expressed in embryos at all stages and mainly in the blastomeres from the 2­cell stage onwards, with the expression concentrated in the nuclei of the blastomeres as identified by strong fluorescence. In the FCF interference group, septin2 was weakly expressed in the nuclei of blastomeres at the 2­ and 4­cell stages, and in the granulated blastomeres at the 4­ and 8­cell stages. Expression was barely observed in and following the morula. Granulation was observed starting from the 4­ and 8­cell stages. Compared with the control group, the FCF interference group exhibited irregular microtubules, abnormal spindle morphology and disordered chromosome arrangement in the blastocysts. The septin2 protein was expressed throughout the early stage embryo from the 2­cell stage to the blastocyst and localized in the nuclei of blastomeres. When the septin protein experienced interference by the FCF inhibitor, septin2 protein expression was reduced, which simultaneously resulted in abnormal embryonic development, uneven cytoplasmic division, various sizes and a reduced number of blastomeres, granulation in the blastomeres, disordered blastocyst microtubule distribution, spindle shape alterations and an abnormality of chromosome arrangement.


Assuntos
Embrião de Mamíferos/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Septinas/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/citologia , Blastômeros/metabolismo , Núcleo Celular/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Camundongos , Microscopia de Fluorescência , Septinas/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
15.
Biochim Biophys Acta Mol Cell Res ; 1866(7): 1230-1238, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30826333

RESUMO

Life begins with calcium. It is the language that a sperm cell uses to respond to instructions from the female reproductive tract to alter its swimming pattern and gain the force required to penetrate the outer layers of the oocyte. The first heartbeat transpires from spontaneous calcium oscillations in embryonic cardiomyocytes. The dynamic balance of calcium between auditory hair cells and the fluid they bathe in enables us to hear our first sound, and our interpretation and response to this sound requires rapid calcium flux through neuronal voltage-sensitive calcium channels. Calcium signaling can decode and integrate informational cues from both the chemical and mechanical cellular microenvironment to drive the form and function of many mammalian organ-systems. Here, we highlight roles for the intracellular calcium signal in the reproductive- and developmental- biology of mammals. A greater appreciation of the signaling pathways that initiate and support life has wide-ranging significance for the fields of reproductive science, neonatology and regenerative medicine. Furthermore, as developmental programs are often reactivated in cancer, an improved understanding of the signaling pathways that underpin mammalian development has important implications for cancer research.


Assuntos
Sinalização do Cálcio , Embrião de Mamíferos/metabolismo , Miócitos Cardíacos/metabolismo , Neoplasias/metabolismo , Oócitos/metabolismo , Reprodução , Espermatozoides/metabolismo , Animais , Embrião de Mamíferos/patologia , Feminino , Humanos , Masculino , Miócitos Cardíacos/patologia , Neoplasias/patologia , Oócitos/patologia , Espermatozoides/patologia
17.
Methods Mol Biol ; 1922: 151-159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838573

RESUMO

Whole-mount in situ hybridization (WMISH) is a commonly used technique for visualizing the expression profile of mRNAs in embryos. Unlike traditional in situ hybridization techniques, which require thin tissue sections, the WMISH technique allows gene expression patterns to be assessed over the entire embryo and structure. Here, we describe the detailed procedural steps of WMISH, including probe production, embryo fixation and staining, and post-hybridization signal detection. Using this protocol, we visualized highly specific expression patterns of Sonic hedgehog and Bmp4 mRNAs in E12.5 mouse embryos.


Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , RNA Mensageiro/análise , Animais , Proteína Morfogenética Óssea 4/genética , Digoxigenina , Desenvolvimento Embrionário/genética , Proteínas Hedgehog/genética , Camundongos , Sondas RNA
18.
Methods Mol Biol ; 1922: 163-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838574

RESUMO

In situ hybridization is a commonly used technique using an antisense RNA probe to localize a specific RNA sequence on histological sections. This approach can visualize the expression pattern of a gene of interest in a portion of tissues. Here, we detail an optimized method for performing in situ hybridization on mouse paraffin sections using digoxigenin (DIG)-labeled RNA probes.


Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , RNA Mensageiro/análise , Animais , Digoxigenina , Camundongos , Inclusão em Parafina , Sondas RNA
19.
Int J Mol Sci ; 20(6)2019 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-30884872

RESUMO

An increasing number of publications indicate that babies born after IVF (in vitro fertilization) procedures have higher rates of anomalies related to imprinting/epigenetic changes, which may be attributed to suboptimal culture conditions. Appropriate maintenance of DNA methylation during the first few days of an in vitro culture requires a supply of methyl donors, which are lacking in current in vitro culture systems. The absence of protection against oxidative stress in the culture increases the risks for errors in methylation. A decrease in the methylation processes is sometimes observed immediately post fertilization, due to delays that occur during the maternal⁻zygotic transition period. Care should be exercised in ART (assisted reproductive technology) procedures in order to avoid the risk of generating errors in methylation during the in vitro culture period immediately post fertilization, which has an impact on imprinting/epigenetics. Formulation of IVF culture media needs to be re-assessed in the perspective of current knowledge regarding embryo physiology.


Assuntos
Metilação de DNA , Embrião de Mamíferos/metabolismo , Epigênese Genética , Fertilização In Vitro , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/anormalidades , Fertilização In Vitro/efeitos adversos , Fertilização In Vitro/métodos , Impressão Genômica , Humanos , Estresse Oxidativo , Zigoto/citologia , Zigoto/metabolismo
20.
Reproduction ; 157(4): 399-411, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30763281

RESUMO

Successful bovine pregnancy establishment hinges on conceptus elongation, a key reproductive phenomenon coinciding with the period during which most pregnancies fail. Elongation is yet to be recapitulated in vitro, whereas in vivo it is directly driven by uterine secretions and indirectly influenced by prior circulating progesterone levels. To better understand the microenvironment evolved to facilitate this fundamental developmental event, uterine fluid was recovered on Days 12-14 of the oestrous cycle - the window of conceptus elongation initiation - from cycling heifers supplemented, or not, with progesterone. Subsequent lipidomic profiling of uterine luminal fluid by advanced high-throughput metabolomics revealed the consistent presence of 75 metabolites, of which 47% were intricately linked to membrane biogenesis, and with seven displaying a day by progesterone interaction (P ≤ 0.05). Four metabolic pathways were correspondingly enriched according to day and P4 - i.e. comprised metabolites whose concentrations differed between groups (normal vs high P4) at different times (Days 12 vs 13 vs 14). These were inositol, phospholipid, glycerolipid and primary bile acid metabolism. Moreover, P4 elevated total uterine luminal fluid lipid content on Day 14 (P < 0.0001) relative to all other comparisons. The data combined suggest that maternal lipid supply during the elongation-initiation window is primarily geared towards conceptus membrane biogenesis. In summary, progesterone supplementation alters the lipidomic profile of bovine uterine fluid during the period of conceptus elongation initiation.


Assuntos
Embrião de Mamíferos/metabolismo , Ciclo Estral/metabolismo , Lipídeos/análise , Metaboloma , Progesterona/farmacologia , Útero/metabolismo , Animais , Bovinos , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Ciclo Estral/efeitos dos fármacos , Feminino , Gravidez , Útero/efeitos dos fármacos
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