Bovine abnormal preimplantation embryos: analysis of segregated cells occurring in the subzonal space and/or blastocoele cavity for their nuclear morphology and persistence of RNA synthesis

Zygote ◽  
2002 ◽  
Vol 10 (2) ◽  
pp. 141-147 ◽  
Author(s):  
J. Pivko ◽  
P. Grafanau ◽  
E. Kubovičová
Keyword(s):  
Rna Synthesis ◽  
Actinomycin D ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Transcription Inhibition ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Labelling Intensity ◽  
Intense Labelling

Bovine embryos in the early blastocyst/blastocyst stage were analysed by [5-3H]uridine labelling followed by electron microscopic autoradiography. In normal control embryos an intact zona pellucida, evenly developed blastomeres and a transparent perivitelline space were seen. In this group, the blastomeres of the trophoblast and embryoblast showed high homogeneous labelling localised in the nucleoplasm and even more intense labelling in the nucleolus. On the contrary, in addition to evident cytoplasmic disintegration, a clearly different labelling pattern and a low labelling intensity were observed in the nuclei of the segregated cells in the subzonal space and in those free in the blastocoele cavity. A typical nuclear morphological feature of these blastomeres was chromatin marginalisation, similar to that observed in embryos treated with actinomycin D for transcription inhibition. It is concluded that the segregated cells are arrested in their further differentiation.

scholarly journals ELECTRON MICROSCOPIC EXAMINATION OF THE SITES OF NUCLEAR RNA SYNTHESIS DURING AMPHIBIAN EMBRYOGENESIS

1965 ◽  
Vol 26 (3) ◽  
pp. 937-958 ◽  
Author(s):  
Shuichi Karasaki
Keyword(s):  
Rna Synthesis ◽  
Developmental Stages ◽  
Early Embryo ◽  
Tail Bud ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Nuclear Rna Synthesis ◽  
Nuclear Rna ◽  
Chromatin Material ◽  
Labeled Rna

The site of H3-uridine incorporation and the fate of labeled RNA during early embryo-genesis of the newt Triturus pyrrhogaster were studied with electron microscopic autoradiography. Isolated ectodermal and mesodermal tissues from the embryos were treated in H3-uridine for 3 hours and cultured in cold solution for various periods before fixation with OsO4 and embedding in Epon. At the blastula stage, the only structural component of the nucleus seen in electron micrographs is a mass of chromatin fibrils. At the early gastrula stage, the primary nucleoli originate as small dense fibrous bodies within the chromatin material. These dense fibrous nucleoli enlarge during successive developmental stages by the acquisition of granular components 150 A in diameter, which form a layer around them. Simultaneously larger granules (300 to 500 A) appear in the chromatin, and they fill the interchromatin spaces by the tail bud stage. Autoradiographic examination has demonstrated that nuclear RNA synthesis takes place in both the nucleolus and the chromatin, with the former consistently showing more label per unit area than the latter. When changes in the distribution pattern of radioactivity were studied 3 to 24 hours after immersion in isotope at each developmental stage, the following results were obtained. Labeled RNA is first localized in the fibrous region of the nucleolus and in the peripheral region of chromatin material. After longer culture in non-radioactive medium, labeled materials also appear in the granular region of the nucleolus and in the interchromatin areas. Further incubation gives labeling in cytoplasm.

High-resolution autoradiographic localization of DNA-containing sites and RNA synthesis in developing nucleoli of human preimplantation embryos: a new concept of embryonic nucleologenesis

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 777-791 ◽  
Author(s):  
J. Tesarik ◽  
V. Kopecny ◽  
M. Plachot ◽  
J. Mandelbaum
Keyword(s):  
Rna Synthesis ◽  
Preimplantation Embryos ◽  
Synthetic Activity ◽  
Rrna Synthesis ◽  
Nucleolar Organizer Regions ◽  
Careful Study ◽  
Matrix Elements ◽  
Electron Microscopic Autoradiography ◽  
General Validity ◽  
Granular Component

Human embryos from the 2-cell to the morula stage, obtained by in vitro fertilization, were incubated with [3H]thymidine or [3H]uridine so as to achieve labelling of all replicating nuclear DNA and the newly synthesized RNA, respectively. The label was localized in different structural components of developing nucleoli using electron microscopic autoradiography. Careful study of the relationship between the structural pattern and nucleic acid distribution made it possible to define four stages of embryonic nucleologenesis. Homogeneous nuclear precursors (i) consist of nucleolar matrix elements appearing as filaments of 3 nm thickness, (ii) do not contain recently replicated DNA and (iii) lack RNA synthetic activity. Penetration of DNA into these bodies is a key event leading to their transformation into heterogeneous nucleolar precursors. In addition to the 3 nm matrix filaments, two types of 5 nm fibrillar components can be recognized in them. The denser type contains DNA and is the site of nucleolar RNA synthesis, while the more loosely arranged 5 nm fibrils are not labelled with [3H]thymidine and apparently represent the newly produced pre-rRNA detached from the transcribing rDNA filament. Compact fibrillogranular nucleoli are characterized by the first appearance of the granular component and reduction of the nontranscribing part of the fibrillar component, both indicating the activation of the machinery for rRNA processing. Finally, the granular component is most evident in reticulated nucleoli, occupying mostly the inner parts of their nucleolonema, while the transcription sites tend to be located at the nucleolar periphery. Our findings advocate a unique concept of embryonic nucleologenesis, different from any other nucleolar event during the cell cycle of differentiated cells. This developmental pattern is characterized by a gradual activation of rRNA synthesis and processing, mediated by progressive association of rDNA and, later on, the newly formed pre-rRNA with pre-existing nucleolar matrix elements that are originally topically separated from nucleolar organizer regions. This model may have a general validity in early animal embryos despite some interspecies variability in the timing of individual steps and resulting structural peculiarities.

scholarly journals 153EXPRESSION OF TGF-BETA I AND TYPE I AND TYPE II OF TGF-BETA RECEPTORS IN BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
B.K. Kim ◽  
H.J. Chung ◽  
B.C. Yang ◽  
D.H. Kim ◽  
J.H. Woo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Type I ◽  
Bovine Embryo ◽  
Type Ii ◽  
Bovine Embryos ◽  
Mrna And Protein Expression ◽  
Bovine Oocytes

Although the effects of TGFβ1, as an important factor in the mice embryo development have been reported, little information relevant to this subject is known in the bovine embryo. The objectives of this study were to investigate the presence and expression patterns of TGFβ1 and TGFβ1 receptors, types I and II, in unfertilized oocytes and fertilized bovine embryos in normal and NT embryo development. We postulated that TGFβ1 may have a beneficial effect on the preimplantation embryo and show different expression patterns at different stages of bovine embryo development. Immature bovine oocytes were aspirated from follicles of ovaries obtained from a local abattoir and they were cultured for up to 24h and fertilized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the presence of TGFβ1 and type I and type II of TGFβ1 receptors (the essential components of the TGFβ1 signaling pathway) in unfertilized oocytes and preimplantation embryos. Also, mRNA and protein expression patterns of TGFβ1 and their receptors at various stages of embryos were examined. It was found that both receptors, as well as TGFβ1, were present in the unfertilized bovine oocytes, indicating that TGFβ1 is a maternally expressed protein. Although the type I TGFβ1 receptor was present at the morulae and blastocyst stages, the type II TGFβ1 receptor was not present at both stages. It was also confirmed that the expression level of TGFβ1 was high at the 8-cell stage, and mRNA and protein expression patterns of TGFβ1 and their receptors were not coincident. Interestingly, TGFβ1 protein was not detected at blastocyst stage of embryos, whereas the mRNA expression level was high at this stage. The results of this experiment indicate that TGFβ1 protein may be needed by embryos after the blastocyst stage and may be expressed in hatched embryos for implantation. These findings support the hypothesis that there may be an interaction between the TGFβ1 and TGFβ1 receptors in the unfertilized oocytes and preimplantation embryos, and that TGFβ1 signaling may be important for the development of the oocytes and the preimplantation embryos.

190 SUPPRESSION OF A TRANSCRIPTION FACTOR MSX1 GENE IN BOVINE PREIMPLANTATION EMBRYOS AND ITS EFFECT ON mRNA, PROTEIN, AND EXPRESSION OF DOWNSTREAM GENES

2008 ◽  
Vol 20 (1) ◽  
pp. 174
Author(s):  
D. Tesfaye ◽  
A. Regassa ◽  
M. Hoelker ◽  
F. Rings ◽  
C. Phatsara ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Growth ◽  
Protein Expression ◽  
Cdna Array ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Expression Levels ◽  
Treatment Groups ◽  
Zygote Stage

MSX1 is a transcription factor gene that orchestrates gene expression and regulates cell growth, proliferation, differentiation, cell-to-cell communication, and the apoptotic pathway during pattern formation in vertebrate embryogenesis. However, its role in bovine preimplantation embryo is not known. Here we aim to investigate the effects of suppressing MSX1 transcript on the development of in vitro-produced bovine embryos, study the expression of mRNA and protein products of the gene, and identify downstream genes using microarray analysis. In the first experiment, IVP zygotes were injected with 341 bp-long dsRNA (LdsRNA) (n = 384), 19 bp small interfering RNA (siRNA) (n = 374), and scrambled sequence RNA (scRNA) (n = 388). Uninjected zygotes (n = 313) were used as control. Developmental phenotype data were collected during culture until Day 8. The mRNA and protein expression levels of the different treatment groups were validated at the 8-cell and blastocyst stages using quantitative real-time PCR and immunohistochemistry, respectively. Developmental phenotype and mRNA data were analyzed using ANOVA under statistical package SPSS (SPSS, Inc., Chicago, IL, USA). In the second experiment, custom SMARTpool siRNA (Dharmacon Inc., Chicago, IL, USA) targeting bovine MSX1 (NM_174798) was used for microinjection together with siRNA and uninjected control. Following treatment at zygote stage, 8-cell embryos were used for mRNA isolation and subsequent array hybridization using bovine cDNA array containing 2000 clones. Array data analysis was performed using statistical analysis of microarray (SAM) procedure. While 33% and 29% of the zygotes from the control and scRNA treatment groups, respectively, reached blastocyst stage, only 20% and 19% of the zygotes from the LdsRNA and siRNA treatment groups, respectively, reached the same stage. Injection of LdsRNA and siRNA at the zygote stage reduced the mRNA expression level by 52% and 33% at the 8-cell stage and by 77% and 87%, respectively, at the blastocyst stage as compared to the control. Similarly, cellular protein expression levels in LdsRNA- and siRNA-injected treatment groups were found to be lower than the control groups at each stage. In all cases, injection of scRNA had no effect on mRNA and protein levels. SAM analysis revealed that, of the total 2000 clones, 3.5% and 5.4% were found to be differentially expressed in embryos injected with SMARTpool and siRNA, respectively, compared to the control. Genes involved in various activities including transcription factors (ALF), cell growth (BMP-15), metabolism (RIOK3), and cytokinasis (AURKA) were found to be down-regulated in 8-cell embryos treated with SMARTpool siRNA compared to the controls. On the other hand, genes involved in protein synthesis (RPL23), energy metabolism (COQ1), cell growth (MNS1) and skeletal development (LGALS3) were found to be upregulated in the same samples. In conclusion, suppression of MSX1 at the mRNA and protein level significantly affected the development of bovine embryos, and our study revealed list of downstream genes regulated by the activity of MSX1.

scholarly journals LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

1967 ◽  
Vol 33 (3) ◽  
pp. 489-496 ◽  
Author(s):  
J. C. H. de Man ◽  
N. J. A. Noorduyn
Keyword(s):  
Orotic Acid ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Electron Microscopic ◽  
Granular Component ◽  
Hepatic Cells ◽  
Progressive Loss ◽  
Nucleolar Rna ◽  
Silver Grains ◽  
Electron Microscopic Radioautography

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.

Electron-microscopic autoradiography of RNA synthesis in the injured myocardium

1977 ◽  
Vol 83 (6) ◽  
pp. 895-898
Author(s):  
V. N. Galankin ◽  
A. A. Pal'tsyn ◽  
A. K. Badikova
Keyword(s):  
Rna Synthesis ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Injured Myocardium

Nucleolus in apoptosis-induced mouse preimplantation embryos

Zygote ◽  
2003 ◽  
Vol 11 (3) ◽  
pp. 271-283 ◽  
Author(s):  
V. Baran ◽  
D. Fabian ◽  
P. Rehak ◽  
J. Koppel
Keyword(s):  
Culture Medium ◽  
Actinomycin D ◽  
Cell Loss ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Rrna Synthesis ◽  
Cell Stage ◽  
Developmental Potential ◽  
Embryonic Genome ◽  
Early Embryos

Apoptosis may occur in early embryos in which the execution of essential developmental events has failed. Thus the initiation of the apoptotic mechanism may be related to activation of the embryonic genome. In this way, developmentally incompetent cells or whole embryos are eliminated. It is likely that some link exists between failed resumption of rRNA synthesis and the incidence of apoptosis in cleaving embryos. In this context, decreased developmental potential in cleaving nucleotransferred embryos is consistent with cell loss, and very likely due to programmed cell death. The effects of apoptosis inducers on cleaving embryos have not been characterised in comparable detail to that in the case of somatic cells. Early embryos provide a very good model for study of these processes because of the specificity of rRNA transcription resumption after fertilization. In our experiments three apoptosis inducers (staurosporin 10 mM, actinomycin D 0.05 mg/ml and camptothecin 0.1 mg/ml) were used in a culture medium for 15 h at the 4-cell stage (day 2) of mouse embryos, followed by further development in a pure culture medium until fixation on days 3, 4 and 5. In staurosporin-induced embryos, light microscopy immunostaining of nucleolar proteins (fibrillarin, Nopp140, protein B23) did not reveal changes in nucleolar morphology on day 3. On days 4 and 5, more compact (roundish) nucleoli (in comparison with controls) were observed. The embryos treated with camptothecin displayed a similar staining pattern to those with staurosporin at each day. In actinomycin-D-treated embryos, marked changes in nucleolar appearance were visible as early as day 3. These changes in nucleolar morphology consisted of loss of the reticulation appearance and fragmentation of nucleoli. In addition to nucleolar changes, significantly decreased cell proliferation was observed. The induced embryos did not reach the blastocyst stage. The number of blastomeres was decreased, and staining with Hoechst 33342 revealed a significant percentage of apoptotic nuclei (condensed/fragmented nuclei) from day 4.

Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 485-493 ◽  
Author(s):  
A.F. Pereira ◽  
L.M. Melo ◽  
V.J.F. Freitas ◽  
D.F. Salamone
Keyword(s):  
Dna Damage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Dna Breaks ◽  
Embryo Production ◽  
Cell Stage ◽  
Γh2ax Foci

SummaryIn vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

Light and electron microscopic autoradiography of coxsackievirus A1 infected muscles: Viral RNA synthesis and inhibition of host cell RNA synthesis

1972 ◽  
Vol 39 (1-3) ◽  
pp. 35-47 ◽  
Author(s):  
K. Bienz ◽  
Denise Egger ◽  
Gertrud Bienz-Isler ◽  
H. Loeffler
Keyword(s):  
Host Cell ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography

Localization of Gallium-67 in Peritoneal Cells by Electron Microscopic Autoradiography

1973 ◽  
Vol 31 ◽  
pp. 404-405
Author(s):  
D. C. Swartzendruber ◽  
Norma L. Idoyaga-Vargas
Keyword(s):  
Clinical Trials ◽  
Soft Tissue ◽  
Tumor Tissue ◽  
Soft Tissue Tumors ◽  
Clinical Usefulness ◽  
Electron Microscopic ◽  
Normal Cells ◽  
Electron Microscopic Autoradiography ◽  
Peritoneal Cells ◽  
Gallium 67

The radionuclide gallium-67 (67Ga) localizes preferentially but not specifically in many human and experimental soft-tissue tumors. Because of this localization, 67Ga is used in clinical trials to detect humar. cancers by external scintiscanning methods. However, the fact that 67Ga does not localize specifically in tumors requires for its eventual clinical usefulness a fuller understanding of the mechanisms that control its deposition in both malignant and normal cells. We have previously reported that 67Ga localizes in lysosomal-like bodies, notably, although not exclusively, in macrophages of the spocytaneous AKR thymoma. Further studies on the uptake of 67Ga by macrophages are needed to determine whether there are factors related to malignancy that might alter the localization of 67Ga in these cells and thus provide clues to discovering the mechanism of 67Ga localization in tumor tissue.

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