• Asian-Australasian Journal of Animal Sciences
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• v.15 no.5
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• pp.615-621
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• 2002

We investigated the effects of the mitogen supplements of 3 types, pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (ConA), to a whole blood culture system on the number of metaphase spreads obtained in perinatal bovine chromosome analysis. In addition, the supplementation of ${\beta}$-mercaptoethanol (${\beta}$-ME) and FBS was examined in such system. Significant differences (p<0.05) were seen in the number of metaphase spreads with PHA stimulation compared to both PWM and ConA stimulation. When examined the effects of ${\beta}$-ME supplementation, the number of metaphase spreads was significantly (p<0.05) increased at $30{\mu}M$ ${\beta}$-ME compared to control. When evaluated FBS supplementation during PWM stimulation, no significant effect of the supplementation was found. Finally, the effects of the cortisol concentration (10-20, 20-30 and >30 ng/ml) of the blood samples were examined. There was no significant effect of cortisol concentration (p>0.05) among these 3 cortisol concentration groups. The mean percentages of normal metaphase plates (2n=60) from each calf 1) with ${\beta}$-ME, 2) without ${\beta}$-ME and 3) with FBS stimulated with PWM were not significantly different (p>0.05). In conclusion, these findings may be useful in cytogenetic screening programs for not only perinatal calves but also for mature cattle.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.375-383
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• 2000

The nuclear maturation and developmental competence of immature, oocytes collected from donors at various morphology of corpus luteum (CL) and fertilized in vitro was investigated by comparing the meiotic activity and the yields of embryos. Ovaries were divided and classified into 4 groups as the following criteria : Group 1 ; ovaries showed evidence of recent ovulation (corpus hemorragicum). Group 2 ; apex of CL was red or brown. Vasculization was limited to periphery of CL. Group 3 ; apex of CL was orange or tan. Vasculization was covered over apex of CL. Group 4 ; CL was light yellow to white and firm in texture and the vascular network on the surface of CL had disappeared. Modified TCM 199 was used for maturation in vitro of immature oocytes and development was induced by using TLP-PVA as a basic medium. When oocytes collected from each group of donors had been matured for 4, 14, and 24 hours in vitro, the proportion of oocytes reaching metaphase I and metaphase II were not different among oocytes from 4 group of ovaries. Mature metaphase II stage of oocytes in each group was first observed at 14 hours, whereas completion of maturation of. oocytes in each group was at 24 hours. Luteal morphology of ovaries had little effect on the proportion of embryos reached 2 cells and 8 cell stage. However, the proportion of embryos cleaved to morula and blastocyst stage was significantly higher in the oocytes obtained from group 1 and 3 than in the oocytes from group 2 and 4 (p＜0.05). This data suggest that reproductive status of the donor significantly influence the yield of in vitro embryos.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.1
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• pp.15-21
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• 2017

Objective: The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of $Ca^{2+}$ chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). Methods: MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a $Ca^{2+}$ chelator to investigate the effect of $Ca^{2+}$ oscillations on their maturation. Results: As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the $Ca^{2+}$ chelator-treated group. Conclusion: The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by $Ca^{2+}$ chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular $Ca^{2+}$ oscillations driven by fertilization.

• Proceedings of the Korean Society of Crop Science Conference
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• 1999.05a
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• pp.162-163
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• 1999

The goal of this research was (1) to describe the conditions and parameters required for the cell cycle synchronization and the accumulation of large number of metaphase cells in maize and other cereal root tips, (2) to isolate intact metaphase chromosomes from root tips suitable for characterization by flow cytometry, and (3) to construct chromosome-specific libraries from maize. Plant metaphase chromosomes have been successfully synchronized and isolated from many cereal root-tips. DNA synthesis inhibitor (hydroxyurea) was used to synchronize cell cycle, follwed by treatement with trifluralin to accumulate metaphase chromosomes. Maize flow karyotypes show substantial variation among inbred lines. thish variation should be sueful in isolating individual chromosome types. In addition, flow cytometry is a useful method to measure DNA content of individual chromosomes in a genotyps, and to detect chromosomal variations. Individual chromosome peaks have been sorted from the maize hybrid B73/Mol7. Libraries were generated form the DOP-PCR amplification product from each peak. To date, we have analyzed clones from a library constructed from the maize chromosome 1 peak. Hybridization of labeled genomic DNA to clone inserts indicated that $24\%,\;18\%,\;and\;58\%$ of the clones were highly repetitive, medium repetitive, and low copy, respectively. Fifty percent of putative low cpoy clones showed single bands on inbred screening, blots, and the remaining $50\%$ were low copy repeats. Single copy clones showing polymorphism will be mapped using recombinant inbred mapping populations. Repetitive clones are being characterized by Southern blot analysis, and will be screened by in situ hybridization for their potential utility as chromosome specific markers.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.357-366
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• 1996

The present experiments aimed to investigate the metabolism of calcium during oocyte maturation in rat. The concentration of free calcium and calmodulin in oocytes was measured respectively by using of fluo-3/AM and FITC with microscope fluorescence spectrometer. The ultrastructural localization of calcium precipitates in oocytes was observed with the transmission electron microscope. Cumulus-free immature oocytes(GV-oocyte) were cultured in vitro through 15 hours. The free calcium concentration in GV oocyte was $55.9{\pm}3.5nM$. In calcium-containing medium, the free calcium concentration was increased in germinal vesicle breakdown(GVBD) oocyte($64.2{\pm}7.3nM$). In normal medium after calcium chelator treatment ($10{\mu}M$ BAPTA/AM), the free calcium contents were slightly lower than those in control group. In calcium-free medium, the free calcium content was drastically increased in GVBD($72.7{\pm}3.4nM$) and metaphase I - anaphase I ($88.0{\pm}3.4nM$) oocyte. In maturation rate of oocytes, GVBD rate was high in control group($82.9{\pm}6.55%$) and calcium chelator treatment group($91.2{\pm}4.4%$), but in calcium-free medium group, it was low and then the oocyte was degenerated without polar body formation. Relative content of calmodulin in oocyte was significantly(P<0.001) increased in metaphase I - anaphase I than in GV and GVBD oocyte. The calcium precipitates were observed in mitochondria and cytoplasm of GV oocyte but that were not observed in mitochondria of GVBD and metaphase I - anaphase I oocyte. And then the calcium precipitates reappeared in mitochondria of metaphase II oocyte. The above results indicate that changes in free calcium and calmodulin concentration of oocyte occur according to the maturational stages and the extracellular calcium is required during oocyte maturation. Also change of calcium localization in oocyte occurs according to the maturational stages.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.71-76
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• 2007

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM This is approximately 500$\sim$1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (${\sim}50{\mu}M$) and butyrolactone (${\sim}100{\mu}M$). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone HI kinase and MAP kinase in mouse oocytes.

• ALGAE
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• v.25 no.4
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• pp.197-204
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• 2010

Cytoskeletal changes were observed during cell division of the green alga Zygnema cruciatum using flourescein isothiocynate (FITC)-conjugated phallacidin for F-actin staining and FITC-anti-$\alpha$-tubulin for microtubule staining. Z. cruciatum was uninucleate with two star-shaped chloroplasts. Nuclear division and cell plate formation occurred prior to chloroplast division. Actin filaments appeared on the chromosome and nuclear surface during prophase, and the F-actin ring appeared as the cleavage furrow developed. FITC-phallacidin revealed that actin filaments were attached to the chromosomes during metaphase. The F-actin ring disappeared at late metaphase. At telophase, FITC-phallacidin staining of actin filaments disappeared. FITC-anti-$\alpha$-tubulin staining revealed that microtubules were arranged beneath the protoplasm during interphase and then localized on the nuclear region at prophase, and that the mitotic spindle was formed during metaphase. The microtubules appeared between dividing chloroplasts. The results indicate that a coordination of actin filaments and microtubules might be necessary for nuclear division and chromosome movement in Z. cruciatum.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.301-305
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• 2006

The aim of this study was to investigate whether addition of porcine epididymal fluid (pEF) into culture medium during in vitro maturation influences the nuclear maturation of porcine germinal vesicle (GV) oocytes. Porcine cumulus-oocyte complexes (COCs) from follicles were cultured in tissue culture medium 199 (TCM 199) containing pEF. After 48hr of culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II (M II) stage was significantly (p<0.05) increased in oocytes cultured in the media supplemented with 10% pEF during in vitro maturation than in those without pEF regardless of cumulus presence or absence (54.6% vs 22.5%, 51.7% vs 24.2%). The supplementation of pEF during maturation of oocyte enhanced oocytes maturation in a dose-dependent manner in vitro. Also significant differences (p<0.05) in the percentage of MII oocytes were observed according to exposure period in pEF. Present study suggests that pEF contains a enhancing component(s) for nuclear maturation of porcine immature oocytes in vitro.

• The Korean Journal of Malacology
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• v.3 no.1
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• pp.24-34
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• 1987

The melaniid snails belonging to genus Semisulcospira were collected in the Kangwha and Yonchon areas of Korea in 1986 through 1987 in order to carry out a cytotaxonomic study, The snails were first narcotized with menthol and fixed with 70% ethyl alcohol for morphological identification. The gonads of adult snails were used for chromosome analyses by the technique of Imai et al. (1977) with minor modification. Slide preparations were observed under high power fields using a Leitz light miscroscope. The results obtained in the present stuedy are summarized as follows: 1)The sanils collected from Kangwha and Yonchon areas were identified as Semisulcospira forticosta(Martens, 1886)and S. gottschei (Martens, 1886) respectively.2)No specific differences were obwerved in details of the chromosome cycle between S. forticosta and S. gottschei.3) Diploid chromosome numbers observed at mitotic metaphase were 36. There was no difference in chromosome numbers between S. forticosta and S. gottschei.4) There were morphological differences in the karyotypes of the two species. The spermatogonial metaphase karyotype of S. forticosta consists of six pairs of metacentric, eleven pairs of submetacentric, and one pair of acrocentric chromosomes. The spermatogonial metaphase karyotype of S. gottschei consists of five pairs of metacentric, tselve pairs of submetacentric, and one pair of acrocentric chromosomes. Summarizing the aboxe results, the two species of Semisulcospira employed in this study have same chromosome numbers(2n=36)with different karyotypes.

• The Korean Journal of Nuclear Medicine
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• v.34 no.1
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• pp.74-81
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• 2000

Purpose: The purpose of this study was to develop in vivo dosimetries using both chromosomal aberrations and micronuclei in mice to assess biological effects of radiations. Materials and Methods: Five each mice were irradiated with 0, 1, 2, 3, 4, 5, 10 Gy of Cs-137 gamma-rays. We scored numbers of chromosomal aberrations in metaphase spreads and numbers of micronuclei in bone marrow smears under light microscope, and obtained the dose-response relationships. We also examined the relationship between the two dose-response curves. Results: The frequency of both chromosomal aberrations and micronuclei increased with dose, in a linear-quadratic manner The delta, beta, and alpha coefficients were 0.0176, 0.0324, and 0.0567 for metaphase analysis (r=1.0, p<0.001) and 0.0019, 0.0073, and 0.0506 for micronuclei assay (r=1.0, p<0.001). The frequency of chromosomal aberrations and micronuclei in different radiation doses was significantly correlated (r=0.99, p<0.01). Conclusion: In vivo dosimetry using either metaphase analysis or micronucleus assay was feasible in mice. These methods could be useful to evaluate biological effects of radiation.