108 CALYCULIN-A INDUCES CHROMOSOME CONDENSATION FOR CYTOGENETIC ANALYSIS IN BOVINE AND MURINE BLASTOMERES

2008 ◽  
Vol 20 (1) ◽  
pp. 134
Author(s):  
J. M. Kramer ◽  
A. Evans ◽  
K. Drury ◽  
K. Moore
Keyword(s):  
Cytogenetic Analysis ◽  
Chromatin Condensation ◽  
Chromosomal Rearrangements ◽  
Chromosome Condensation ◽  
Preimplantation Embryos ◽  
Hypotonic Solution ◽  
Calyculin A ◽  
Chi Square ◽  
Degree Of Condensation

Cytogenetic studies of preimplantation embryos have traditionally used interphase fluorescence in situ hybridization (FISH) to examine chromosome copy number, chromosomal rearrangements, or sex determination. Comprehensive analysis of chromosomes is hindered by the low mitotic index of blastomeres, combined with the technical limitations of interphase FISH. Efforts to overcome these limitations include inducing premature chromosome condensation (PCC) for metaphase FISH, by fusing blastomeres with metaphase II-stage oocytes, or with time-consuming incubations with okadaic acid or vinblastine. Our objective was to evaluate Calyculin-A (CA) as an alternative to induce PCC in blastomeres. In vitro-produced bovine 8-cell embryos and frozen–thawed mouse 8-cell embryos were cultured with CA and examined for changes in morphology before fixation. Colcemid (0.1 mg mL–1), the standard for cytogenetic analysis, and vehicle served as positive and negative controls. Blastomeres were washed in hypotonic solution, loaded onto slides, and fixed in methanol:acetic acid (3:1). The degree of chromatin condensation and quality of chromosome spreads were determined by 42,6-diamidino-2-phenylindole staining and visualization on an epifluorescence microscope (100�). Experiment 1 (1 rep, 136 cells) tested dose of CA on bovine blastomeres at 50, 100, 150, 250, 500, and 750 nm. Experiments 2 and 3 tested culture duration of CA (50 nm) at 60, 120, and 180 min in bovine blastomeres and 60, 90, and 120 min in mouse blastomeres (2 reps each, 132 and 207 cells, respectively). Data were analyzed by chi-square with significance deemed P < 0.05. Cell lysis and blebbing was observed in bovine blastomeres treated with greater than 150 nm CA. Duration of CA treatment affected the frequency of bovine and mouse blastomeres undergoing PCC. Fewer bovine blastomeres treated for 60 min underwent chromatin condensation compared to blastomeres treated for at least 120 min with 50 nm CA (25% v. 100%; P < 0.05). In mice, the frequency of blastomeres undergoing PCC was lower (43%) for 50 nm CA at 60 min than at 90 and 120 min (90 and 100%, respectively; P < 0.05). Chromatin condensation suitable for cytogenetic analysis was 20, 34, and 20%, respectively, but did not differ (P > 0.10). Further examination (Exp. 4, 5 reps, 293 cells) comparing the degree of condensation revealed 55% of the total bovine blastomeres treated with 50 nm CA for 120 min were suitable for cytogenetic analysis, as compared to 30% treated with colcemid for 16 h (P < 0.05). Bovine and mouse blastomeres treated with vehicle had lower PCC than all other treatments averaging 2 and 7%, respectively (P < 0.05). These results suggest that CA can rapidly induce PCC in blastomeres from pre-implantation bovine and murine embryos, but the degree of chromatin condensation may not always be suitable for detailed cytogenetic analysis from a single blastomere.

Cytogenetic analysis of human preimplantation embryos following developmental arrest in vitro

1998 ◽  
Vol 10 (6) ◽  
pp. 505 ◽  
Author(s):  
Paula A. Almeida ◽  
Virginia N. Bolton
Keyword(s):  
Chromosomal Abnormality ◽  
Cytogenetic Analysis ◽  
Chromosomal Abnormalities ◽  
Preimplantation Embryo ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Developmental Arrest ◽  
The Relationship ◽  
Human Preimplantation Embryos

The relationship between chromosomal abnormalities in the human preimplantation embryo and developmental arrest in vitro was investigated. Cytogenetic analysis of 171 embryos that had arrested between the pronucleate and the 8-cell stages demonstrated that the overall incidence of chromosomal abnormality among these embryos was 63.4%. Of the embryos that arrested at the pronucleate stage (n = 48), 47.9% were chromosomally abnormal, compared with 59.5% of those that arrested between the 2- and 4-cell stages (n = 50), and 82.8% of those arrested between the 5- and 8-cell stage (n = 73). The rate of abnormality in embryos with poor morphology (irregular shaped blastomeres and considerable extracellular fragmentation) was significantly higher (86.8%; n = 33) than those with good morphology (60%; n = 51; P<0.005). These results suggest that there is an association between chromosomal abnormality, developmental arrest in vitro, and poor morphology.

scholarly journals Interphase Cytogenetic Analysis of Micronucleated and Multinucleated Cells Supports the Premature Chromosome Condensation Hypothesis as the Mechanistic Origin of Chromothripsis

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1123 ◽  
Author(s):  
Pantelias ◽  
Karachristou ◽  
Georgakilas ◽  
Terzoudi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cytogenetic Analysis ◽  
Alternative Hypothesis ◽  
Chromatin Condensation ◽  
Human Lymphocytes ◽  
Chinese Hamster ◽  
Chromosome Condensation ◽  
Premature Chromosome Condensation ◽  
Multinucleated Cells

The discovery of chromothripsis in cancer genomes challenges the long-standing concept of carcinogenesis as the result of progressive genetic events. Despite recent advances in describing chromothripsis, its mechanistic origin remains elusive. The prevailing conception is that it arises from a massive accumulation of fragmented DNA inside micronuclei (MN), whose defective nuclear envelope ruptures or leads to aberrant DNA replication, before main nuclei enter mitosis. An alternative hypothesis is that the premature chromosome condensation (PCC) dynamics in asynchronous micronucleated cells underlie chromosome shattering in a single catastrophic event, a hallmark of chromothripsis. Specifically, when main nuclei enter mitosis, premature chromatin condensation provokes the shattering of chromosomes entrapped inside MN, if they are still undergoing DNA replication. To test this hypothesis, the agent RO-3306, a selective ATP-competitive inhibitor of CDK1 that promotes cell cycle arrest at the G2/M boundary, was used in this study to control the degree of cell cycle asynchrony between main nuclei and MN. By delaying the entrance of main nuclei into mitosis, additional time was allowed for the completion of DNA replication and duplication of chromosomes inside MN. We performed interphase cytogenetic analysis using asynchronous micronucleated cells generated by exposure of human lymphocytes to γ-rays, and heterophasic multinucleated Chinese hamster ovary (CHO) cells generated by cell fusion procedures. Our results demonstrate that the PCC dynamics during asynchronous mitosis in micronucleated or multinucleated cells are an important determinant of chromosome shattering and may underlie the mechanistic origin of chromothripsis.

scholarly journals Introduction into in vitro culture and cytogenetic analysis of Iris attica Boiss. & Heldr. and Iris pseudopumila Tineo plants

2019 ◽  
Vol 16 (2) ◽  
pp. 203-211 ◽  
Author(s):  
M. O. Twardovska ◽  
I. O. Andreev ◽  
V. A. Kunakh
Keyword(s):  
Chromosome Number ◽  
In Vitro Culture ◽  
Cytogenetic Analysis ◽  
Root Meristem ◽  
Chromosomal Rearrangements ◽  
High Survival ◽  
High Survival Rate ◽  
Survival Level

Aim. The work was aimed at the development of conditions for introduction into in vitro culture of two species of irises, Iris attica and I. pseudopumila to obtain aseptic seedlings with subsequent reintroduction into natural environment, as well as at cytogenetic analysis of the obtained plants. Methods. In vitro seed germination and seedling cultivation. Cytogenetic analysis of cells of root meristem, determination of chromosome number and morphology in mitotic metaphase plates, anaphase analysis. Results. The plants of I. attica and I. pseudopumila were introduced in vitro. Aseptic seedlings were obtained, which were actively growing on MS/2 medium without phytohormones. The experiments on the adaptation of the plants to greenhouse conditions revealed the high survival rate for both species. The chromosome number 2n = 16 was established for the obtained plants of both I. attica and I. pseudopumila. Mixoploidy was detected in root meristem of some of the plants, the incidence of which was 10.9 % for I. pseudopumila and 30.4 % for I. attica. The frequency of cells with chromosomal rearrangements revealed by anaphase analysis in root meristem of I. pseudopumila seedlings was 2.6 %; in I. attica plants, chromosome aberrations were not detected. Conclusions. The plants of I. attica and I. pseudopumila were introduced into in vitro culture, aseptic seedlings were obtained, which showed a high survival level when adapted to greenhouse conditions. Chromosome number 2n = 16 was established for the obtained plants of both species. The root apical meristems of the seedlings were found to be mixoploid, with the incidence of mixoploidy in I. attica identified as three times higher than in I. pseudopumila plants.Keywords: I. attica Boiss. & Heldr., I. pseudopumila Tineo, aseptic seedlings, mixoploidy, anaphase aberration.

The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes

Development ◽  
2002 ◽  
Vol 129 (7) ◽  
pp. 1715-1727 ◽  
Author(s):  
Silvia Di Agostino ◽  
Pellegrino Rossi ◽  
Raffaele Geremia ◽  
Claudio Sette
Keyword(s):  
Okadaic Acid ◽  
Mapk Pathway ◽  
Chromatin Condensation ◽  
Active Role ◽  
Mek Inhibitor ◽  
Chromosome Condensation ◽  
Cell Extracts ◽  
Pachytene Spermatocytes

Chromosome condensation during the G2/M progression of mouse pachytene spermatocytes induced by the phosphatase inhibitor okadaic acid (OA) requires the activation of the MAPK Erk1. In many cell systems, p90Rsks are the main effectors of Erk1/2 function. We have identified p90Rsk2 as the isoform that is specifically expressed in mouse spermatocytes and have shown that it is activated during the OA-triggered meiotic G2/M progression. By using the MEK inhibitor U0126, we have demonstrated that activation of p90Rsk2 during meiotic progression requires activation of the MAPK pathway. Immunofluorescence analysis indicates that activated Erks and p90Rsk2 are tightly associated with condensed chromosomes during the G2/M transition in meiotic cells. We also found that active p90Rsk2 was able to phosphorylate histone H3 at Ser10 in vitro, but that the activation of the Erk1/p90Rsk2 pathway was not necessary for phosphorylation of H3 in vivo. Furthermore, phosphorylation of H3 was not sufficient to cause condensation of meiotic chromosomes in mouse spermatocytes. Other proteins known to associate with chromatin may represent effectors of Erk1 and p90Rsk2 during chromosome condensation. Nek2 (NIMA-related kinase 2), which associates with chromosomes, plays an active role in chromatin condensation and is stimulated by treatment of pachytene spermatocytes with okadaic acid. We show that inhibition of the MAPK pathway by preincubation of spermatocytes with U0126 suppresses Nek2 activation, and that incubation of spermatocyte cell extracts with activated p90Rsk2 causes stimulation of Nek2 kinase activity. Furthermore, we show that the Nek2 kinase domain is a substrate for p90Rsk2 phosphorylation in vitro. These data establish a connection between the Erk1/p90Rsk2 pathway, Nek2 activation and chromosome condensation during the G2/M transition of the first meiotic prophase.

The experience of holding the cycles of assisted reproductive technology with defrost, a biopsy, genetic study and refreezing of embryos in patients with multiple unsuccessful implantations

2016 ◽  
pp. 166-170
Author(s):  
Y.V. Masliy ◽  
◽  
I.O. Sudoma ◽  
P.S. Mazur ◽  
D.A. Mykytenko ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Genetic Diagnosis ◽  
Morphological Characteristics ◽  
Implantation Rate ◽  
Ovarian Response ◽  
Reproductive Technologies ◽  
Comparative Genome Hybridization ◽  
Comparative Genome ◽  
Vitro Fertilization

The objective: to study the possibility of using frozen blastocysts for biopsy and genetic testing and performance measurement transfer euploeded 5–7-day-old embryos after thawing, biopsies, refreezing and thawing in patients with unsuccessful implantation. Patients and methods. The object of the study was the group of patients with repeated failure of implantation (4) in programs of auxiliary reproductive technologies (ART), subject to transfer to the uterus in total (i.e. in all the programs) for at least 6 good quality embryos based on morphological characteristics). All women had sufficient ovarian reserve. The patient was treated for infertility within the ART programs of the clinic of reproductive medicine "Nadiya" in the period from 2006 to 2016. The sample included couples who were not carriers of chromosomal rearrangements, without anomalies of the uterus (congenital and acquired: a doubling of the uterus, one-horned uterus, intrauterine membrane, synechia, submucous myoma of the uterus). All women had a positive ovarian response to controlled stimulation with gonadotropins (at least 7 oocytes) and a sufficient number of cryopreserved embryos. The first group (G1) included 64 women who trophectodermal a biopsy was performed on fresh blastocysts (in a loop controlled ovarian hyperstimulation). The second group (G2) were included 31 women who underwent thawing previously cryopreserved blastocysts trophectodermal re-biopsy and vitrification of blastocysts. Results. It was found that the performance of transfers euploid embryos that were vitrified, bioptrone and revitriphted, a little lower than those that were bioptrone fresh and vitrified only once. At the same time computationa genetic diagnosis previously vitrified blastocysts using comparative genome hybridization in patients with recurrent failed implantation allows to obtain a reasonable pregnancy rate (58%), implantation rate (33.3 %) and the birth of living children (45.1 %). Conclusion. Reprising biopropane embryos does not cause significant destructive impact and allows you to achieve pregnancy and birth of the alive child. Key words: in vitro fertilization, reusable unsuccessful implantation, a method of comparative genome hybridization, refreezing.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals Comparison of bacterial microleakage of three bioactive endodontic sealers in simulated underwater diving and aviation conditions

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mehdi Dastorani ◽  
Behnam Malekpour ◽  
Mohsen AminSobhani ◽  
Mohammadsadegh Alemrajabi ◽  
Arezoo Mahdian ◽  
...  
Keyword(s):  
Atmospheric Pressure ◽  
Mineral Trioxide Aggregate ◽  
In Vitro Study ◽  
Chi Square ◽  
Custom Made ◽  
Single Cone ◽  
Registration Number ◽  
Chi Square Test ◽  
Pressure Changes

Abstract Background Bacterial microleakage is an important cause of apical periodontitis and endodontic treatment failure. This study aimed to assess the bacterial microleakage of nano-mineral trioxide aggregate (nano-MTA) as a sealer, Endoseal MTA, and GuttaFlow Bioseal sealers in atmospheric pressure, and simulated underwater diving and aviation conditions. Methods In this in vitro, experimental study, 180 extracted single-rooted teeth were cleaned and shaped, and were then randomly divided into three groups for single-cone obturation using Endoseal MTA, GuttaFlow Bioseal, or nano-MTA as a sealer. Each group was then randomly divided into three subgroups, and subjected to ambient atmospheric pressure, 2 atm pressure (to simulate underwater diving), and 0.5 atm pressure (to simulate aviation) using a custom-made pressure chamber. The teeth then underwent microbial leakage test using Streptococcus mutans (S. mutans), and the percentage of samples showing microleakage was recorded for up to 1 month, and analyzed using the Chi-square test. Results The three sealer groups were significantly different regarding bacterial microleakage (P < 0.05). The nano-MTA group showed significantly higher microleakage after 15 days than the other two groups (P = 0.006). The effect of pressure on bacterial microleakage was not significant in any sealer group (P > 0.05). Conclusion Within the limitations of this in vitro study, it may be concluded that single-cone obturation technique using nano-MTA as a sealer results in lower resistance to bacterial microleakage compared with the use of GuttaFlow Bioseal, and Endoseal MTA. Pressure changes in simulated underwater diving and aviation conditions had no significant effect on bacterial microleakage. Trial Registration Number This is not a human subject research.

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

2004 ◽  
Vol 287 (4) ◽  
pp. H1730-H1739 ◽  
Author(s):  
Ron Zohar ◽  
Baoqian Zhu ◽  
Peter Liu ◽  
Jaro Sodek ◽  
C. A. McCulloch
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cardiac Fibroblasts ◽  
Chromatin Condensation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Wild Type ◽  
Nuclear Fragmentation ◽  
A Cell

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN−/−) mice were treated in vitro with H2O2to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN−/−cells but was increased to only 20% in WT cells. In contrast, after 1–8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN−/−cells. Electron microscopy of WT cells treated with H2O2showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN−/−cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN−/−and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN−/−cells by ∼30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN−/−cells was not altered. Restoration of OPN expression in OPN−/−fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

Sign in / Sign up

Export Citation Format

Share Document