163 SUCROSE ADDITION AT EARLY CULTURE STAGE IMPROVES DEVELOPMENT OF PRE-IMPLANTATION PORCINE NT AND IVF EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 189
Author(s):  
I.-S. Hwang ◽  
J.-S. Seo ◽  
H.-S. Park ◽  
S.-W. Kim ◽  
D.-H. Kim ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Formation Rate ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Culture Stage ◽  
Membrane Changes ◽  
Analysis System

Apoptosis is a form of cell death leading to fragmentation of the DNA, shrinkage of the cytoplasm, membrane changes, and cell death without lysis or damage to neighboring cells. It might contribute to the low developmental rate of in vitro-produced (IVP) embryos, but apoptosis in porcine embryos is still unclear. This study investigated the effect of sucrose in the culture medium on the development of porcine NT and IVP embryos. Oocytes were aspirated from the follicles in ovaries collected from a local abattoir, and then matured in TCM-199 for 40-44 h. Fresh semen was diluted and equilibrated at 16�C. A final concentration of motile spermatozoa was adjusted to 5 � 105 cells/mL in fertilization medium. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus and used as donor cells. Embryos were cultured in PZM-3 supplemented with 0.05 M sucrose for 2 days, and then cultured in the PZM-3 without sucrose for 4 days at 38.5�C under 5% CO2 in air. Apoptotic cell death was analyzed by using a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. All data were subjected to a generalized linear model procedure (PROC-GLM) of the statistical analysis system (SAS; SAS Institute, Inc., Cary, NC, USA). NT and IVF embryos cultured in the medium supplemented with sucrose showed a significantly higher blastocyst formation rate than those cultured with no addition (28.8 vs. 20% and 32.3 vs. 17.9%; P < 0.05, respectively). For apoptosis, both NT and IVF embryos cultured in the medium with sucrose showed significantly lower frequency of apoptosis compared to embryos cultured in the medium without sucrose (3.4 vs. 6.3% and 0.6 vs. 1.8%; P < 0.05, respectively). Finally, the number of nuclei in NT blastocysts cultured in the medium with sucrose was higher than that of NT blastocysts cultured without sucrose (30.8 vs. 25.5; P < 0.05, respectively). However, the number of nuclei in the IVF blastocysts was not significantly different between groups. These results indicate that sucrose addition may increase the development of porcine NT and IVF embryos to the blastocyst stage and decrease the rate of apoptotic cells.

scholarly journals In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2234-2243 ◽  
Author(s):  
Giorgio Zauli ◽  
Marco Vitale ◽  
Elisabetta Falcieri ◽  
Davide Gibellini ◽  
Alessandra Bassini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Apoptotic Cell ◽  
High Surface ◽  
Suspension Cultures ◽  
Apoptotic Cell Death ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Terminal Phase ◽  
Serum Free

Abstract To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.

scholarly journals In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2234-2243 ◽  
Author(s):  
Giorgio Zauli ◽  
Marco Vitale ◽  
Elisabetta Falcieri ◽  
Davide Gibellini ◽  
Alessandra Bassini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Apoptotic Cell ◽  
High Surface ◽  
Suspension Cultures ◽  
Apoptotic Cell Death ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Terminal Phase ◽  
Serum Free

To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.

115 APOPTOSIS EVENT OF PREIMPLANTATION DEVELOPMENT STAGES IN PORCINE IN VITRO-FERTILIZED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 157
Author(s):  
S. M. Hong ◽  
S. H. Jeong ◽  
S. H. Hyun
Keyword(s):  
Cell Death ◽  
Actinomycin D ◽  
Apoptotic Cell ◽  
Formation Rate ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Cell Stage ◽  
Development Stages

Little is known about apoptosis events in porcine preimplantation embryos. In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro-fertilized (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22 h postinsemination were treated at different concentrations of actinomycin D (0, 5, 50 and 500 ng mL–1 in NCSU medium). Four groups were incubated at 37°C in 5% CO2, 5%O2 for 8 h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2 days and BL development rate at 7 days after in vitro culture (IVC). A significantly less rate of cleavage was found in the 500 ng mL–1 group compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 15.4% v. 48.6%, 40%, 32%). In the results of BL formation rate, there was a significantly less BL formation rate in 500 ng mL–1 compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 0% v. 10%, 8.8%, 9%). In experiment 2, to evaluate apoptotic cells at different stage (2-cell, 4-cell, 8-cell and BL stage) of all groups, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng mL–1 actinomycin D). A high number of the BL derived control group contained at least one apoptotic cell. Actinomycin D treated BL responded to the presence of apoptotic inductor by a significant decrease in the average number of blastomeres and a significant increase in the incidence of apoptotic cell death. In the 500 ng mL–1 group, the incidence of apoptosis increased at the 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is a useful tool for the apoptosis study of porcine preimplantation embryos. This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.

Apoptotic processes and DNA cytosine methylation in mouse embryos arrested at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 269-279 ◽  
Author(s):  
Dušan Fabian ◽  
Alexandra Bukovská ◽  
Štefan Juhás ◽  
Juraj Koppel
Keyword(s):  
Cell Death ◽  
Cytosine Methylation ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Blastocyst Stage ◽  
Nuclear Condensation ◽  
Cell Stage ◽  
Genome Methylation ◽  
Dna Cytosine Methylation

SummaryThe present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (α-amanitin, actinomycin D, TNF-α). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.

scholarly journals KUS121 attenuates the progression of monosodium iodoacetate-induced osteoarthritis in rats

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sachiko Iwai ◽  
Hanako O. Ikeda ◽  
Hisashi Mera ◽  
Kohei Nishitani ◽  
Motoo Saito ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Apoptotic Cell ◽  
Intraperitoneal Administration ◽  
Atp Depletion ◽  
Knee Joints ◽  
Cellular Protection ◽  
Monosodium Iodoacetate

AbstractCurrently there is no effective treatment available for osteoarthritis (OA). We have recently developed Kyoto University Substances (KUSs), ATPase inhibitors specific for valosin-containing protein (VCP), as a novel class of medicine for cellular protection. KUSs suppressed intracellular ATP depletion, endoplasmic reticulum (ER) stress, and cell death. In this study, we investigated the effects of KUS121 on chondrocyte cell death. In cultured chondrocytes differentiated from ATDC5 cells, KUS121 suppressed the decline in ATP levels and apoptotic cell death under stress conditions induced by TNFα. KUS121 ameliorated TNFα-induced reduction of gene expression in chondrocytes, such as Sox9 and Col2α. KUS121 also suppressed ER stress and cell death in chondrocytes under tunicamycin load. Furthermore, intraperitoneal administration of KUS121 in vivo suppressed chondrocyte loss and proteoglycan reduction in knee joints of a monosodium iodoacetate-induced OA rat model. Moreover, intra-articular administration of KUS121 more prominently reduced the apoptosis of the affected chondrocytes. These results demonstrate that KUS121 protects chondrocytes from stress-induced cell death in vitro and in vivo, and indicate that KUS121 is a promising novel therapeutic agent to prevent the progression of OA.

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.

scholarly journals AKT is involved in granulosa cell autophagy regulation via mTOR signaling during rat follicular development and atresia

Reproduction ◽  
2014 ◽  
Vol 147 (1) ◽  
pp. 73-80 ◽  
Author(s):  
JongYeob Choi ◽  
MinWha Jo ◽  
EunYoung Lee ◽  
DooSeok Choi
Keyword(s):  
Cell Death ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Apoptotic Cell ◽  
Follicular Development ◽  
Negative Regulator ◽  
Metabolic Clearance ◽  
Akt Inhibitor ◽  
Western Blots

In this study, we examined whether granulosa cell autophagy during follicular development and atresia was regulated by the class I phosphoinositide-3 kinase/protein kinase B (AKT) pathway, which is known to control the activity of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Ovaries and granulosa cells were obtained using an established gonadotropin-primed immature rat model that induces follicular development and atresia. Autophagy was evaluated by measuring the expression level of microtubule-associated protein light chain 3-II (LC3-II) using western blots and immunohistochemistry. The activity of AKT and mTOR was also examined by observing the phosphorylation of AKT and ribosomal protein S6 kinase (S6K) respectively. After gonadotropin injection, LC3-II expression was suppressed and phosphorylation of AKT and S6K increased in rat granulosa cells. By contrast, gonadotropin withdrawal by metabolic clearance promoted LC3-II expression and decreased phosphorylation of AKT and S6K. In addition,in-vitroFSH treatment of rat granulosa cells also indicated inhibition of LC3-II expression accompanied by a marked increase in phosphorylation of AKT and S6K. Inhibition of AKT phosphorylation using AKT inhibitor VIII suppressed FSH-mediated phosphorylation of S6K, followed by an increase in LC3-II expression. Furthermore, co-treatment with FSH and AKT inhibitor increased the levels of apoptosis and cell death of granulosa cells compared with the single treatment with FSH. Taken together, our findings indicated that AKT-mediated activation of mTOR suppresses granulosa cell autophagy during follicular development and is involved in the regulation of apoptotic cell death.

Biocatalytic synthesis of terpene esters and their biological activity in human glioma cells

2021 ◽  
Vol 22 ◽  
Author(s):  
Mateusz Kutyła ◽  
Aleksandra Maciejczyk ◽  
Mariusz Trytek ◽  
Joanna Jakubowicz-Gil
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Fold Increase ◽  
Point Of View ◽  
Terpene Alcohols ◽  
Human Glioblastoma Multiforme ◽  
Anti Cancer ◽  
Therapeutic Molecules ◽  
Resistance To Chemotherapy

Background: Gliomas are highly malignant brain tumors with high resistance to chemotherapy. Therefore, investigations of new therapeutic molecules with high anti-glioma activity are of great importance. Objective: In this work, biocatalytic esterification of terpene alcohols with proven anti-cancer activity was performed to enhance their potency to induce cell death in human glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cell lines in vitro. Method and Results: We used primary terpene alcohols and carboxylic acids with a length of two to nine carbon atoms. The structure of the drinks influenced the esterification efficiency, which decreased in the following order: monocyclic > linear > bicyclic. Terpene alcohols and their esters only induced apoptotic cell death, which is highly desirable from a therapeutic point of view but did not induce autophagy and necrosis. The esterification of perillyl alcohol with butyric acid caused a 4-fold increase in cell death induction in the T98G line. Citronellol valerate, caprylate, and pelargonate and myrtenol butyrate, caprylate, and pelargonate also showed higher activity than their alcohol precursors. Conclusion: We have herein shown that esterification of natural alcohols by biocatalysis can improve the activity for other compounds investigated for their anti-glioma activity.

scholarly journals Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase.

1994 ◽  
Vol 180 (4) ◽  
pp. 1547-1552 ◽  
Author(s):  
M G Cifone ◽  
R De Maria ◽  
P Roncaioli ◽  
M R Rippo ◽  
M Azuma ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Apoptotic Cell ◽  
Human Tumor ◽  
Apoptotic Cell Death ◽  
Cell Surface Receptor ◽  
Cross Linking ◽  
Cell Extracts

Intracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.

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