Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Development ◽  
1988 ◽  
Vol 104 (3) ◽  
pp. 453-463
Author(s):  
A.P. Dyban ◽  
K. Lee ◽  
G.T. O'Neill ◽  
S. Speirs ◽  
M.H. Kaufman
Keyword(s):  
Silver Nitrate ◽  
Silver Staining ◽  
Cytogenetic Study ◽  
Metaphase Plate ◽  
Rrna Synthesis ◽  
Cell Nuclei ◽  
Interphase Nuclei ◽  
Cell Stage ◽  
Nucleolar Activity ◽  
Silver Nitrate Staining

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.

The effect of double-strength standard saline citrate on silver staining. I. Nucleoli and micronucleoli in the somatic and germ line of the grasshopper Arcyptera fusca (Orthoptera)

1986 ◽  
Vol 28 (2) ◽  
pp. 219-226 ◽  
Author(s):  
J. Gosàlvez ◽  
J. de la Torre ◽  
C. Garcia de la Vega ◽  
C. López-Fernández
Keyword(s):  
Silver Nitrate ◽  
Silver Staining ◽  
Germ Line ◽  
Standard Technique ◽  
Previous Treatment ◽  
Nucleolar Activity ◽  
Standard Saline Citrate ◽  
Nucleolar Proteins ◽  
In Cells

The nucleolar activity in cells from both somatic and germ line of the grasshopper Arcyptera fusca has been analyzed by means of silver staining using a standard technique with and without a previous treatment of the slides with double-strength standard saline citrate (2 × SSC). Results show that the treatment with 2 × SSC improves the silver staining of the main nucleoli and in some tissues reliably uncovers other sites along the chromosomes with attached silver precipitates (micronucleoli). The parallel origin of the main nucleoli and the micronucleoli, the possible enlargement of rDNA activity in some tissues, and the ability of the silver nitrate to stain certain nucleolar proteins are discussed.Key words: Arcyptera, Orthoptera, nucleolus, micronucleus, silver staining.

scholarly journals Effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer

Reproduction ◽  
2003 ◽  
pp. 535-542 ◽  
Author(s):  
X Li ◽  
JL Tremoleda ◽  
WR Allen
Keyword(s):  
Embryonic Development ◽  
Nuclear Transfer ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Metaphase Ii Oocytes ◽  
Adult Fibroblasts

The effects of repeated passage in vitro of fetal fibroblast cells (FFC) and adult fibroblast cells (AFC) on nuclear remodelling and first embryonic division when used to reconstruct horse oocytes, and the reasons for the developmental block in progression to the two-cell stage were investigated. A total of 463 metaphase II oocytes produced 427 fibroblast-cytoplasm couplets after nuclear transfer, which finally resulted in 319 reconstructed oocytes. With increasing numbers of passages, the rates of nuclear remodelling decreased in both types of donor cell; about half of the fused donor cell nuclei showed the S-G2-prometaphase stages of the first embryonic division 18-20 h after cell-fusion treatment, irrespective of the number of donor cell passages (FFC: 49%; AFC: 53%). The rates of first embryonic division in the reconstructed oocytes fell with increasing age of the donor cells (FFC: 32%-26%-23%; AFC: 27%-23%-24%) and these rates were significantly lower than those obtained from metaphase II oocytes activated parthenogenetically (79%, P < 0.05). Microscopic analysis of the organization of the first embryonic division in the developmentally blocked oocytes reconstructed with either FFC or AFC showed that most of these (FFC: 78%; AFC: 92%) could not form the mitotic spindle and the metaphase plate of chromosomes. These findings indicate that either fetal or adult fibroblasts that have undergone relatively few passages in vitro are most suitable as donors. However, both types of cell have lower potential to restart first embryonic development after nuclear transfer than do the equivalent cells in other species. Improvement in the rate of donor cell nuclear progression from S-G2-prometaphase to beyond the metaphase stage, and the normal organization of first embryonic development in reconstructed horse oocytes, would seem to be the key to the production of cloned embryos in this species.

scholarly journals 212 HETEROGENEITY OF RIBOSOMAL RNA GENE ACTIVATION AMONG CELLS OF IN VITRO-PRODUCED PORCINE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 256
Author(s):  
B. Bjerregaard ◽  
F. Strejcek ◽  
Z. Rasmussen ◽  
J. Laurincik ◽  
H. Niemann ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Silver Staining ◽  
Transcriptional Activity ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Cell Stage ◽  
Nucleolar Proteins

In vitro production (IVP) of porcine embryos by in vitro maturation of oocytes followed by fertilization and culture in vitro is hampered by great deficiencies. Initiation of at least the major embryonic genome transcription, which includes activation of ribosomal RNA (rRNA) genes and the associated formation of a fibrillo-granular nuclealus, is normally seen during the 4-cell stage in pigs. We have investigated the activation of rRNA synthesis and the presence of silver staining nucleolar proteins in porcine IVP embryos as a marker of transcriptional activity and, thus, developmental competence. A total of 205 porcine IVP embryos from the 2-cell to the blastocyst stage were examined using sequential fluorescent in situ hybridization (FISH) to the rRNA genes and their transcripts and silver staining of nucleolar proteins as previously described (Viuff et al. 2002 Biol. Reprod. 66, 629–634). Briefly, cumulus-oocyte complexes with at least three cumulus cell layers and evenly granulated ooplasm were isolated from 2–5 mm ovarian follicles with stereomicroscopic evaluation. Subsequently, oocytes were matured in NCSU-37 and mechanically denuded followed by fertilization using frozen-thawed epididymal semen. Presumptive zygotes were then cultured in NCSU-23 at 39°C, 5% CO2. Around the time of expected cleavage, the embryos were examined every second hour to determine the time of cleavage. Embryos at the 2-cell stage were harvested at 5 h post-cleavage (hpc), 4-cell embryos late during the third cell cycle at 30 hpc, and tentative 8- and 16-cell embryos at 10 hpc. Blastocysts were harvested at Day 5 post-insemination. In general, nuclei of 2-cell embryos displayed 4 small foci of FITC labelling (presumably the rDNA), but no specific silver staining, and were consequently categorized as transcriptionally inactive. At the late 4-cell stage, 58% of the embryos resembled the 2-cell stage. However, in the remaining embryos (42%), some or all nuclei displayed large areas of FISH labelling (presumptive rDNA and rRNA) co-localized with silver staining, and were catagorized as transcriptionally active. Among the 8-cell embryos, 64% displayed a majority of transcriptionally active nuclei, whereas this was the case in 83% and 92% of the embryos in the 16-cell embryos and the blastocysts, respectively. In general, the majority of the embryos contained a mixture of transcriptionally active and inactive cells. These findings show that the porcine IVP embryos are often delayed and asynchronous with respect to activation of the rRNA genes. Table 1. Categorization of nuclei according to transcriptional activity This work was supported by grants from “Disease models, disease prevention and animal welfare improvement: The pig embryo as a model.” Danish Research Agency (Grant: 9901178), NATO (Grant: 978658), and Deutsche Forschungsgemeinschaft (DFG).

scholarly journals Karyotype, constitutive heterochromatin and nucleolar organizer regions (NORs) in Belosacris coccineipes (Acrididae-Leptysminae)

2000 ◽  
Vol 23 (3) ◽  
pp. 575-579 ◽  
Author(s):  
Vilma Loreto ◽  
Maria José de Souza
Keyword(s):  
Sex Determination ◽  
Silver Nitrate ◽  
X Chromosome ◽  
Silver Staining ◽  
Constitutive Heterochromatin ◽  
Nucleolar Organizer ◽  
Nucleolar Organizer Regions ◽  
C Banding ◽  
Nucleolar Organizers ◽  
Silver Nitrate Staining

Several techniques including C-banding, fluorochromes and silver staining were used to obtain information about heterochromatin patterns in the grasshopper B. coccineipes. Conventional staining showed a karyotype with 2n = 23 chromosomes in males and 2n = 24 in females, as well as XO:XX sex determination and acrotelocentric chromosomes. The medium-sized X chromosome was heteropycnotic positive at the beginning of prophase I and negative in metaphase I. C-banding revealed heterochromatic blocks in the pericentromeric regions of all chromosomes. Silver nitrate staining in this species showed three small bivalents (S9-S11) as nucleolar organizers with NORs located in the pericentromeric regions. CMA3-positive blocks were seen in pericentromeric regions of pairs M6, S9, S10 and S11. Sequential staining with CMA3/AgNO3 revealed homology between the CMA3-positive bands and NORs of the bivalents S9, S10 and S11. The CMA3-positive block of the bivalent M6 could represent a latent secondary NOR. The results obtained permit us to distinguish two categories of the constitutive heterochromatin in B. coccineipes.

Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1263-1270
Author(s):  
M. Biggiogera ◽  
K. Burki ◽  
S.H. Kaufmann ◽  
J.H. Shaper ◽  
N. Gas ◽  
...  
Keyword(s):  
Silver Staining ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Dense Fibrillar Component ◽  
Cell Stage ◽  
Nucleolar Activity ◽  
Morula Stage ◽  
Protein B23 ◽  
Ultrathin Sections ◽  
Fibrillar Component

The ultrastructural distribution of proteins B23 and nucleolin in the nucleolus of mouse embryos from the zygote to the early blastocyst has been analyzed by means of specific antibodies and immunocytochemistry using colloidal gold complexes as markers. In parallel, silver staining of nucleoli was carried out on ultrathin sections. Our results show that the compact prenucleolar bodies at 1- and 2-cell stage as well as the compact residual fibrillar masses observed up to the morula stage, are labelled with the two antibodies. These masses, however, are not stained with silver up to the 4-cell stage. In well-developed nucleoli, the two antibodies co-localize in the dense fibrillar component (DFC) and the granular component (GC) while fibrillar centers (FCs) are devoid of label. On the contrary, silver staining occurs in the FCs and DFC but not in the GC. Our observations suggest that there is no direct relationship between the occurrence of silver staining and the distribution of protein B23 or nucleolin. Moreover, neither the localization of the two above proteins nor silver staining are unequivocally related to the nucleolar activity.

scholarly journals STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

2021 ◽  
Vol 22 (1) ◽  
pp. 460
Author(s):  
Huan Ou-Yang ◽  
Shinn-Chih Wu ◽  
Li-Ying Sung ◽  
Shiao-Hsuan Yang ◽  
Shang-Hsun Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Promoter Assay ◽  
Maternal To Zygotic Transition ◽  
Mouse Zygotes ◽  
Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Study of DNA integrity in somatic cells and spermatozoa by single-cell gel electrophoresis with silver-nitrate staining

2008 ◽  
Vol 2 (1) ◽  
pp. 87-92
Author(s):  
S. N. Proshin ◽  
G. V. Stepanov ◽  
V. Yu. Kravtsov ◽  
G. P. Kosyakova ◽  
I. A. Paranyan ◽  
...  
Keyword(s):  
Silver Nitrate ◽  
Single Cell ◽  
Gel Electrophoresis ◽  
Somatic Cells ◽  
Dna Integrity ◽  
Single Cell Gel Electrophoresis ◽  
Silver Nitrate Staining

Scanning electron microscopy of the ependymal surface of the third ventricle after silver nitrate staining

1973 ◽  
Vol 61 ◽  
pp. 207-216 ◽  
Author(s):  
J.E. Bruni ◽  
D.G. Montemurro ◽  
R.E. Clattenburg ◽  
R.P. Singh
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Silver Nitrate ◽  
Third Ventricle ◽  
The Third ◽  
Silver Nitrate Staining ◽  
Scanning Electron

Staining SDS-PAGE Gels of Skeletal Matrices after Western Blot: A Way to Improve their Sharpness

2016 ◽  
Vol 672 ◽  
pp. 215-221 ◽  
Author(s):  
Frédéric Marin ◽  
Françoise Immel ◽  
Nolwenn Trinkler ◽  
Danièle Gaspard
Keyword(s):  
Western Blot ◽  
Silver Nitrate ◽  
Sds Page ◽  
Simple Protocol ◽  
Classical Technique ◽  
The Matrix ◽  
Partial Transfer ◽  
Silver Nitrate Staining ◽  
Extra Step ◽  
Calcified Tissues

Denaturing 1D electrophoresis on acrylamide gels, also referred as SDS-PAGE, is a classical technique for fractionating and visualizing the macromolecular constituents of matrices associated to calcified tissues. This technique has been widely used in association with the subsequent silver nitrate staining. But because matrices associated to calcified tissues are very often glycosylated and constituted of numerous polydisperse macromolecules, the obtained pattern is frequently ‘smeary’ and discrete bands, when present on the gel, are often blurred and thickened. In this paper, we present a simple protocol that can circumvent this drawback and ‘clean’ the gels. In short, after the classical migration step of the matrix macromolecules, the gel is electro-blotted on a PVDF membrane, similarly to a Western blot, but for a shorter time (partial transfer, i.e., one hour or less). It is subsequently stained with silver nitrate. The likely effect of the transfer is to partly remove polydisperse macromolecules and to ‘sharpen’ the discrete bands. We think that this extra-step may improve in several cases the gel pictures, particularly when they are blurred. We illustrate this phenomenon with two examples taken from brachiopod and mollusc shell matrices.

Different distributions of homologous chromosomes in adult human Sertoli cells and in lymphocytes signify nuclear differentiation

1996 ◽  
Vol 109 (4) ◽  
pp. 773-776 ◽  
Author(s):  
A.C. Chandley ◽  
R.M. Speed ◽  
A.R. Leitch
Keyword(s):  
Sertoli Cells ◽  
Cell Types ◽  
Fish Analysis ◽  
Cell Nuclei ◽  
Interphase Nuclei ◽  
Homologous Chromosomes ◽  
Blood Lymphocytes ◽  
Painting Probes ◽  
Whole Chromosome Painting

Using whole chromosome painting probes for human chromosomes 3,7,8,13,17 and 21 and X and the probe pHY2.1 for the Y chromosome coupled with fluorescent in situ hybridization (FISH) analysis, the distribution of chromosomes is reported in nuclei of Sertoli cells of the adult testis and in stimulated blood lymphocytes. The distribution of chromosomes in the two cell types is significantly different. A strong tendency for each pair of homologues to pair is inferred from the observation of only a single detectable signal in the majority of Sertoli cell nuclei. The sex chromosomes, by contrast, give two clearly separated signals. Interphase nuclei in dividing blood lymphocytes, analysed as controls, also show mainly two separated signals for all non-acrocentric autosomal pairs, but acrocentric pairs no. 13 and 21 show some tendency to associate, probably reflecting satellite association.

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