• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.75-81
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• 2004

Objective: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. Methods: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1, 2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-$\beta$-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). Results: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. Conclusion: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

• Biomolecules & Therapeutics
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• v.2 no.3
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• pp.286-291
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• 1994

The mutagenicity of DA-3030(rhG-CSF)was studied by reverse mutation test, chromosome aberration test and micronucleus test. The reverse mutatuon test in bacteria was performed using salmonella typhimurium strain TA100, TA98, TA1535 and TA1537 with rhG-CSF in any of the concentrations(150, 75, 37.5, 18.75, 9.375 and 4,6875 $\mu\textrm{g}$/plate), no increase in the number of revertant colonies in each strain was observed, irrespective of treatment with the metabolic activation system(S-9 mix) The chromosome aberration test was carried out using CHL cells, cell line from chinese hamster lung. With 4 doses(75, 37.5, 18.75 and 9.375 $\mu\textrm{g}$/ml) of rhG-/CSF the cells were treated for 24 or 48 hours in the direct method or for 6 hours followed by 18 hour-expression time in the metabolic activation method. Results of the study showed, by the direct method or metabolic activation method, no trend toward increase in the number of aberrant metaphase. The micronucleus test was carried out using ICR mice at the age of 8 weeks. Three doses(862.5, 1725 and 3450 $\mu\textrm{g}$/kg) of DA-3030 were admintstered intraperitoneally with single shot and bone marrow cells were sampled at 24 hours after administration. Neither the number of polychromatic erythrocytes with micronuclei nor the ratio of normochromatic erythrocytes to polychromatic erythrocytes increased singinficantly in each dose, compared with a vehicle control. These results indicate that rhG-CSF has not mutagenic potential under the condiions.

• Journal of Life Science
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• v.8 no.1
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• pp.60-66
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• 1998

The mototic and meiotic chromosomal characteristics of ICR mice were investigated with G-and C-banding techniques. For the puposes, the chromosomal preparations were made with the modified air-drying method of Imal et al. Chromosomal analysis using testis could be observed mitotic as well as meitotic chromosomal behaviors, and the centromeric regions of all chromosomes including X chromosome were strongly stained in C-banded preparations. Nineteen autosomal bivalents and a single uniequal terminally associated X-Y bivalent in normal cells were observed during the late prophase and the metaphase of the meiosis I. The mean frequencies of previously dissociated X-Y chromosomes in the primary apermatocytes of the control group were 7.45%, but the frequencies of X-Y dissociation in the alkylating agents-treated group were about 3-4 times higher than that in the control group. Application of C-banding in meiotic stages could be certainly distinguish between vibalent type and univalents type of sex chromosomes.

• Korean Journal of Medicinal Crop Science
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• v.11 no.5
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• pp.402-407
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• 2003

Karyotype analysis and chromosomal localization of 5S and 45S rDNAs using multi-color fluorescence in situ hybridization (McFISH) technique were carried out in Bupleurum longeradiatum. Somatic metaphase chromosome number was 2n=12. Karyotype was composed of three pairs of metacentrics (No.3, 4 and 6) and three pairs of submetacentrics (No. 1, 2 and 5). The length of somatic prometaphase chromosomes ranges from 2.55 to $5.05{\mu}m$ with total length of $18.15\;{\mu}m$. In FISH experiment, one pair of 5S rDNA signals was detected on the pericentromeric region of chromosome 4 and one pair of 45S rDNA signals was detected on the telomeric region of chromosome 2.

• Korean Journal of Medicinal Crop Science
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• v.13 no.1
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• pp.48-51
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• 2005

Karyotype analysis and chromosomal locailization of 45S and 5S rDNAs using McFISH (multi-color fluorescence in situ hybridization) were carried out in Jeffersonia dubia Benth., which is one of medicinal plants belonging to Berberidaceae. The somatic metaphase chromosome number was 2n=2x=12 and the size of chromosomes ranged $1.95{\sim}3.50\;{\mu}m$. The chromosome complement consisted of two pairs of metacentrics (chromosomes 1 and 3), two pairs of submetacentrics (chromosomes 2 and 4) and two pairs of subtelocentrics (chromosomes 5 and 6). In McFISH, one pair of 45S rDNA site was detected on the centromeric region of chromosome 2 and three pairs of 5S rDNA sites were detected on the short arm of chromosomes 4, 5 and 6, respectively.

• Development and Reproduction
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• v.16 no.1
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• pp.59-67
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• 2012

Previously, we found that $Zap70$ (Zeta-chain-associated protein kinase) expressed in the mouse oocytes and played significant role in completion of meiosis specifically at MI-MII (metaphase I-II) transition. Microinjection of $Zap70$ dsRNA into the cytoplasm of germinal vesicle oocyte resulted in MI arrest, and exhibited abnormalities in their spindles and chromosome configurations. The purpose of this study was to determine the mechanisms of action of $Zap70$ in oocyte maturation by evaluating downstream signal networking after $Zap70$ RNAi (RNA interference). The probe hybridization and data analysis were used by Affymetrix Gene Chip Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Total 1,152 genes were up (n=366) and down (n=786) regulated after $Zap70$ RNAi. Among those genes changed, we confirmed the expressional changes of the genes involved in the regulation of actin cytoskeleton and MAPK (mitogen-activated protein kinase) signaling pathway, since the phenotypes of $Zap70$ RNAi in oocytes were found in the changes in the chromosome separation and spindle structures. We confirmed the changes in gene expression in the actin skeletal system as well as in the MAPK signaling pathway, and concluded that these changes are main cause of the aberrant chromosome arrangement and abnormal spindles after $Zap70$ RNAi.

• Korean Journal of Agricultural Science
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• v.48 no.1
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• pp.163-170
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• 2021

Bacterial infections in the female reproductive tract negatively affect ovarian function, follicular development, and embryo development, leading to the eventual failure of fertilization. Moreover, bacterial lipopolysaccharides (LPS) can interfere with the immune system and reproductive system of the host animal. Therefore, this study examined the effect of LPS on the in vitro maturation (IVM) of pig oocytes. Oocytes were matured in TCM199 medium in the presence of varying concentrations of LPS (0 - 50 ㎍·mL^-1). The maturation rate, cortical granules (CGs) migration, and chromosome alignment were subsequently evaluated during the meiotic development of the oocytes. We observed a dose-dependent and significant decrease in the metaphase II (MII) rate with increasing concentrations of LPS (97.6% control [0 ㎍·mL^-1 LPS] vs. 10.4-74.9% LPS [1 - 50 ㎍·mL^-1], p < 0.05). In addition, compared to the control oocytes without LPS, higher levels of abnormal CGs distribution (18.1 - 50.0% LPS vs. 0% control), chromosome/spindle alignment (20.3 - 56.7% LPS vs. 0% control), and intracellular ROS generation were observed in oocytes matured with LPS (p < 0.05). Nitrite levels were also increased in the maturation medium derived from the oocytes matured with LPS (p < 0.05). These results indicate that LPS induces oxidative stress during IVM and affects oocyte maturation, including CGs migration and chromosome alignment of pig oocytes.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.481-488
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• 2022

Background: Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methods: Cancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. Results: Ginsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. Conclusion: Ginsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.

• Horticultural Science & Technology
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• v.30 no.5
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• pp.568-572
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• 2012

Determination of ploidy level for the mother plant is prerequisite for effective breeding. The study was carried out to determine the ploidy level in 7 different plant materials by FISH karyotype analysis. Among the seven plant varieties analyzed, all exhibit tetraploid (2n = 4x = 28) based on the results observed in chromosome analysis. Four signals of 45S rDNAs were detected on the terminal region of the short arm of chromosome 7. The length of somatic metaphase chromosomes ranges from 1.67 to $2.67{\mu}m$ in 'Alexandra', 1.40 to $2.04{\mu}m$ in 'Freud', 1.64 to 2.24 in 'Little Silver', 1.69 to $2.26{\mu}m$ in 'Teresa', 1.70 to $2.65{\mu}m$ in 'Tineke', 1.35 to $2.08{\mu}m$ in 'Vital', 1.39 to $2.04{\mu}m$ in 'Yellow Mimi'. Total length of the chromosome ranges from $11.23{\mu}m$ in 'Freud' as minimum to $15.05{\mu}m$ in 'Alexandra' as maximum. The karyotypes were composed of metacentric, submetacentric, and subtelocentric chromosome but there is no subtelocentric chromosome.

• Korean Journal of Plant Resources
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• v.20 no.5
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• pp.446-450
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• 2007

Tiarella polyphylla D. Don(Saxifragaceae) is a perennial herb and distributed in China, Japan, Taiwan and Korea. Especially, it only grows in Ulleung island of Korea. It has been using for asthma, bruise and audition troubles with main components of some Triterpenoids and seven oleanolic Saponins. There is only known its chromosomal number rarely and cytogenetic study was not done. From this study, karyotype analysis and chromosomal localization of 5S and 45S rDNAs using bicolor-FISH(fluorescence in situ hybridization) were carried out. The somatic metaphase chromosome number was 2n=2x=14 and the size of chromosomes ranged $1.66{\sim}3.50{\mu}m$. The chromosome complement consisted of four pairs of submetacentrics(chromosomes 1, 2, 3 and 6), two pairs of subtelocentrics(chromosomes 5 and 7) and one pair of telocentrics(chromosome 4). We also observed NOR(nucleolus organizer region) on the chromosome 4. In bicolor-FISH, one pair of 55 and 45S rDNA sites was detected on the centromeric region of chromosome 3 and short arm of chromosome 4, respectively. Bicolor FISH was very useful tool for the localization and identification of rDNAs on the chromosomes in Tiarella polyphylla.