OBJECTIVE: The purpose of this study was to evaluate the effect of CCB treatment on the survivability and in vitro development of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then exposed to EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes on EM grid was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M sucrose at 37degrees C for 3 minutes. This was followed by 0.5 M and 0.25 M sucrose for 3 minutes, each. We compared the survivability, cleavage and developmental rate of mouse frozen by vitrification between CCB treated and non-treated groups. Chi-square was used to determine statistical significance. statistical significance was defined as p<0.05. RESULTS: Survivability (79.3%) and developmental rate into blastocyst (52.3%) of mouse oocyte were markedly decreased after vitrification. There were no significant differences between CCB treated and non- treated groups regarding survivability of oocyte frozen by vitrification (80.3% vs 78.5%). The developmental rate into 2-cell in CCB treated group was significantly higher than that in non-treated group (69.7% vs 61.9%, p<0.05). The developmental rate into blastocyst in CCB treated group was higher than that in non-treated group (54.9% vs 51.5%), but the difference was not significant. CONCLUSION: Survivability of mouse oocyte could not be affected by CCB treatment and developmental rate into 2-cell was improved in CCB treated group. It is suggested that CCB treatment prior vitrification improve stability of cytoskeleton and then improve fertilization and early stage embryo development.
Animals ; Blastocyst ; Cytochalasin B* ; Cytoskeleton ; Embryonic Development ; Female ; Fertilization ; Mice* ; Nitrogen ; Oocytes* ; Pregnancy ; Sucrose ; Vitrification*

OBJECTIVE: The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at 37degress C for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at 4degress C overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. RESULTS: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05).CONCLUSION: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.
Actin Cytoskeleton ; Animals ; Cytochalasin B* ; Cytoskeleton ; Formaldehyde ; Immunoglobulin G ; Mice* ; Microtubules ; Nitrogen ; Oocytes* ; Propidium ; Sucrose ; Vitrification*

OBJECTIVE: The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at 37degress C for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at 4degress C overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. RESULTS: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05).CONCLUSION: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.
Actin Cytoskeleton ; Animals ; Cytochalasin B* ; Cytoskeleton ; Formaldehyde ; Immunoglobulin G ; Mice* ; Microtubules ; Nitrogen ; Oocytes* ; Propidium ; Sucrose ; Vitrification*

The survival rate and motility recovered after cryopreservation of testicular spermatozoa in testicular sperm extraction (TESE)-ICSl program is low. The purpose of this study was to assess the availability and efficiency of mouse empty zona pellucida in cryopreserving human TESE spermatozoa. Mouse empty zonae pellucidae were obtained by extraction of cytoplasm with or without cytochalasin B treatment. Motile sperm from proven-fertile donor and two azoospermic patients after TESE were individually inserted into empty zona pellucida and cryopreserved. Two to five days after cyropreservation, the frozen sperm were thawed and the rates of recovery and motility were observed. The ooplasmic extraction rates of control (N=80) and cytochalasin B treated oocytes (N=80) were 94.0% and 96.2%, respectively (p>0.05). The post-thaw recovery rates of spermatozoa and rates of motility recovery of ejaculate (N=70) and testicular (N=70) sperm were 97.1%, 97.1% and 95.7%, 94.3%, respectively (p>0.05). The results of this study showed that the mouse zone pellucida is useful for cryostorage of single testicular spermatozoa.
Animals ; Cryopreservation* ; Cytochalasin B ; Cytoplasm ; Herpes Zoster* ; Humans ; Mice* ; Oocytes ; Sperm Injections, Intracytoplasmic* ; Spermatozoa* ; Survival Rate ; Tissue Donors ; Zona Pellucida*

The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [3H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [3H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [3H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [3H]3-OMG uptake was increased at 48 h. However, [3H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [3H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [3H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition.
Alendronate ; Animals ; Blood-Retinal Barrier ; Capillaries* ; Cytochalasin B ; Diphosphonates* ; Endothelial Cells* ; Glucose* ; Hand ; Histamine ; Mevalonic Acid ; Rats* ; Retinaldehyde*

PURPOSE: Epiretinal membrane in proliferative vitreoretinopathy (PVR) may cause tractional retinal detachment after vitreoretinal surgery. It has been thought that the proliferative membrane is mainly composed of choroidal fibroblasts and retinal pigment epithelial cells. Inspite of the technical advances, the treatment of PVR is still difficult. Therefore, the need for phamarcologic treatment of proliferative vitreoretinopathy is increasing. METHODS: In vitro models of proliferative vitreoretinopathy allow to identify the factors which may inhibit proliferation and contraction of collagen matrix by choroidal fibroblast and retinal pigment epithelial cells. Cultured choroidal fibroblasts and the RPE cells were plated to the collagen matrix and antiproliferative drugs was tested. RESULTS: Each antiproliferative drug showed the inhibition of collagen matrix contraction at following concentration: colchicine(0.1 microgram/ml), puromycin(1~10 microgram/ml), cytochalasin B(0.05 microgram/ml). Transmission electron micrograph of collagen matrices showed dense collagen fibers surrounding choroidal fibroblast and fine collagen fibers surrounding RPE cell. Scanning electron micrograph of collagen matrices contaning colchicine, puromycin, or cytochalasin B showed that collagen fibers were well preserved without distortion. All collagen matrices containing RPE cells showed more fine collagen fibers than those containing choroidal fibroblasts. CONCLUSION: Colchicine, puromycin, cytochalasin B showed inhibitory effect on cell mediated contraction in addition to potent antiproliferative effect. Retinal pigment epithelial cell played less significant role in causing PVR than choroidal fibroblast.
Choroid* ; Colchicine ; Collagen* ; Cytochalasin B ; Epiretinal Membrane ; Epithelial Cells* ; Fibroblasts* ; Membranes ; Puromycin ; Retinal Detachment ; Retinaldehyde* ; Traction ; Vitreoretinal Surgery ; Vitreoretinopathy, Proliferative

The nucleotide-oligomerization domain (NOD) proteins are members of the NOD-like receptor (NLR) family, which are intracellular and cytoplasmic receptors. We analyzed the role of NODs for cytokine production by macrophages infected with intracellular pathogen M. leprae, the causative agent of leprosy. Production of pro-inflammatory cytokines such as IL-1beta and TNF-alpha was inhibited in the presence of cytochalasin D, an agent blocking phagocytosis, suggesting that intracellular signaling was, partially, required for macrophage activation to M. leprae infection. Next, we investigated the role of NOD1 and NOD2 proteins on NF-kappaB activation and cytokine expression. Treatment with M. leprae significantly increased NF-kappaB activation and expression of TNF-alpha and IL-1beta in NOD1- and NOD2-transfected cells. Interestingly, their activation and expression were inhibited by cytochalasin D, suggesting that stimulation of NOD proteins may be associated with the enhancement of cytokine production in host to M. leprae.
Cytochalasin D ; Cytokines ; Humans ; Leprosy ; Macrophage Activation ; Macrophages ; Mycobacterium ; Mycobacterium leprae ; NF-kappa B ; Phagocytosis ; Proteins ; Receptors, Cytoplasmic and Nuclear ; Tumor Necrosis Factor-alpha

To evaluate the role of capsular polysaccharide (CPS) as a virulence factor, the interaction of V. vulnificus with mouse peritoneal macrophages and serum, which are involved in the clearance of bacteria from blood and other tissues, were examined. In this study, MO6-24/0 (wild strain; hemolysin- and capsule-positive), MO6-24/I' (acapsular spontaneous mutant), CVD 752 (acapsular transposon mutant), and CVD 707 (hemolysin-negative and capsule-positive mutant) were used. The strain with CPS (MO6-24/0 and CVD 707) were more resistant to phagocytosis by mouse peritoneal macrophages compared with acapsular strains (MO6-24/T and CVD 752), and the resistance to phagocytosis was not changed by serum opsonin in the capsular strains. Acapsular strains were more susceptible to serum bactericidal activity than the capsular strains through the classical complement pathway. MO6-24/0 strain were detected in blood, spleen, liver and lung at 4 hours after intraperitoneally infection, whereas CVD 752 were not detected. All tested strains could induced the transcription of inflammatory cytokine gene such as IL-1, IL-6, IL-10 and TNF-u, and their inductions were not decreased by cytochalasin B treatment. This results demonstrate that CPS of V. vulnificus plays an important role in V. vulnificus infection through interfering nonspecific host defense system such as blood clearance and phagocytosis.
Animals ; Bacteria ; Complement Pathway, Classical ; Cytochalasin B ; Interleukin-1 ; Interleukin-10 ; Interleukin-6 ; Liver ; Lung ; Macrophages, Peritoneal ; Mice ; Phagocytosis ; Spleen ; Vibrio vulnificus* ; Vibrio* ; Virulence*

OBJECTIVES: We investigated the genotoxic effects of 40-59 nm silver nanoparticles (Ag-NPs) by bacterial reverse mutation assay (Ames test), in vitro comet assay and micronucleus (MN) assay. In particular, we directly compared the effect of cytochalasin B (cytoB) and rat liver homogenate (S9 mix) in the formation of MN by Ag-NPs. METHODS: Before testing, we confirmed that Ag-NPs were completely dispersed in the experimental medium by sonication (three times in 1 minute) and filtration (0.2 microm pore size filter), and then we measured their size in a zeta potential analyzer. After that the genotoxicity were measured and especially, S9 mix and with and without cytoB were compared one another in MN assay. RESULTS: Ames test using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains revealed that Ag-NPs with or without S9 mix did not display a mutagenic effect. The genotoxicity of Ag-NPs was also evaluated in a mammalian cell system using Chinese hamster ovary cells. The results revealed that Ag-NPs stimulated DNA breakage and MN formation with or without S9 mix in a dose-dependent manner (from 0.01 microg/mL to 10 microg/mL). In particular, MN induction was affected by cytoB. CONCLUSIONS: All of our findings, with the exception of the Ames test results, indicate that Ag-NPs show genotoxic effects in mammalian cell system. In addition, present study suggests the potential error due to use of cytoB in genotoxic test of nanoparticles.
Animals ; Comet Assay ; Cricetinae ; Cricetulus ; Cytochalasin B ; DNA ; Female ; Filtration ; Liver ; Micronucleus Tests ; Mutagenicity Tests* ; Nanoparticles* ; Ovary ; Rats ; Salmonella typhimurium ; Silver* ; Sonication

This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(P<0. 01). The rates of enucleation and cytoplast with diameter over 5min in 34 degrees C group were higher than those in 25 degrees C group (all P<0. 01). The cytoplast purities were (95.43 +/- 0. 59)% in 38% Percoll groups,which were higher than those of 40% Percoll (P<0. 05). Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling. The results further showed that the phenotype of HL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.
Cell Compartmentation ; Cell Nucleus ; Cell Separation ; Centrifugation, Density Gradient ; Colchicine ; analogs & derivatives ; pharmacology ; Cytochalasin B ; pharmacology ; Cytoplasm ; HL-60 Cells ; Humans