• Journal of Animal Reproduction and Biotechnology
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• v.35 no.3
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• pp.223-231
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• 2020

Karyotype analysis is a major work in the process of triploid abalone production for the purpose of productivity and quality improvement. However, the metaphase spreads for karyotype analysis have been prepared just from the larvae at trochophore stage, which has restricted the spectrum of sample correction inhibiting more efficient analysis. Here, we investigated the feasibility of preparing metaphase spreads from the larvae at veliger stage that is the next developmental stage of trochophore. For this, diploid and triploid larvae at trochophore and veliger stages from Pacific abalone (Haliotis discus hannai) were subjected to metaphase spread preparation and its efficiencies were measured and compared each other. As the results, although the efficiencies of metaphase spread preparation were significantly lower in the larvae at veliger stage compared to the ones at trochophore stage regardless of ploidy status, we found that the preparation of metaphase spreads, which showed the clear chromosomal images containing the normal number of chromosomes, was possible from the veliger stage larvae. On the other hands, all larvae used in this study regardless of developmental stage and ploidy did not show colchicine sensitivity. Moreover, no significant difference was observed in cell cycle distribution of the cells comprising larvae between two developmental stages regardless of ploidy status. These suggested that the details of protocol to prepare metaphase spreads from abalone larvae should be optimized depending on its developmental stages. Taken together, we demonstrated the feasibility of preparing metaphase spreads from H. discus hannai veliger stage larvae for karyotype analysis.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.2
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• pp.111-118
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• 2003

Objectives: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. Materials and Methods: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. Results: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were $1.0{\mu}M$ of vinblastine (20.3%), $5.0{\mu}M$ of nocodazole (28.1%) and $1.0{\mu}M$ colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine ($1.0{\mu}M$) and nocodazole ($1.0{\mu}M$). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. Conclusions: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.

• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.70-78
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• 1989

Mouse eggs recovered from oviducts at one hourly intervals between 10 and 20 hours after administration of hCG were fixed, stained and then investigated the rate of in vitro fertilization and nuclear maturation. In case of out- bred ICR mice, ovulations were occured between 11 and 13 hours after hCG injection. The stages of in vitro maturation of eggs recovered from female mice at various times after hCG injection were metaphase I, anaphase I, telophase I and metaphase II. However the majority was metaphase I(17.6 to 44.4%) and metaphase II(42.9 to 80.0%) stage. When the eggs were inseminated with epididymal spermatozoa, the fertilization rate was declined as the egg recovery time after hCG administration was delayed. That is, the proportion of eggs undergoing fertilization became higher(68.1 to 77.4%) in the eggs at 12 to 15hr after injection of hCG than those(17.5 to 56.4) at 16 to 20 hr after injection of hCG. Also, when nuclear maturation of the unfertilized eggs were observed at 8 hours after insemination, the majority was in metaphase I and metaphase II and no anaphase I and telophase I were observed.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.2
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• pp.3-9
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• 1964

Root tips of wheat, soybean, cotton and barley were treated with cold temperature (12 or 24 hrs in $0^{\circ}C$ or 2$^{\circ}C$), 8-Hyd.exyquinolin (2 or 10 hrs in 0.03 or 0.1%) and colcllicine (2 or 10 hrs in 0.2 or 1, 0%), and the frequency of metaphase were observed. The results were summarized as follow; 1. Chilling the seminal roots before or after sooting from the seed significantly increased tile number of mitotic cells and the rate of metaphase cells to mitotic cells. The optimal duration of chilling seemed to be differ depending on the kinds of plant and 24 hours to be too long except wheat so far examined here. 9. 8-Hydroxyquinolin treatment, about 2 hours in 0.03%, increased the rate of metaphase cells. The higher concentration and the longer treatment of this chemical caused the lower frequency of mitotic cells generally. 3. Colchicine treatment, 2 to 10 hrs in 0.2 to 1.0%, increased the frequency of mitotic cells and the rate of metaphase cells. Colchicine treatment was same or superior than any other treatments on the increase of metaphase cells. 4. All the treatments examined here caused chromosome contraction with most serious in colchicine following by 8-Hydroxyquinolin and chilling. 5. The feasibility of general application of chilling under the check of proper temperature and proper duration depending on the kinds of crops were discussed.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.235-240
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• 2004

In vitro maturation of denuded porcine immature oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. This study was to determine whether the addition of spermatozoa into the culture medium could influence the nuclear maturation of porcine cumulus-enclosed germinal vesicle (GV) oocytes. Cumulus-oocyte complexes (COCs) were collected from follicles of 3- to 5-mm diameter. Porcine COCs were cultured in tissue culture medium containing spermatozoa. After 48 h 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 was significantly (P < 0.05) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa (52.3% vs 12.5%). No difference in the percentage of metaphase II was observed among the different periods of spermatozoa exposure and among the spermatozoa from different species. The proportion of oocytes reaching metaphase II was significantly different between high and low concentrations of spermatozoa. The present study suggests that manunalian spermatozoa contain a substance(s) that improves nuclear in vitro maturation of porcine cumulus-enclosed GV oocytes. Enhancing effect of spermatozoa for in vitro maturation of oocytes is a highly dose-dependent.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.95-101
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• 1996

The present study investigated the effect of treatment with nocodazole on the efficiency of cell cycle synchronization and development of mouse 4-cefl embryos. In addition, developmental ability of reconstituted embryos that received nuclei from 4-cell embryos synchronized with nocodazole at M phase was examined in vitro and in vivo. (1) When 4-cell blastorneres exposed to culture medium containing l$\mu$g /ml nocodazole for 2, 4, 6 and 8 hrs, 40% (40/10l), 75% (l19/159), 85% (87/102) and 97% (155/160) of nuclei were synchronized at M phase, respectively. (2) Treated with nocodazole for 4 hrs, the proportion of 4-cell embryos developed to blastocysts (98%, 60/61) was not significanUy different from that of the control embryos (98%, 196/201). However, the developmental ability of 4-cell embryos treated for 8 (87%, P<0.05)and 12 hrs (76%, P

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.41-41
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• 2001

Chromatin configuration and microtubule assembly were determined in porcine maturing and activated oocytes following intracytoplasmic sperm injection. Microtubule localization was confirmed using a mouse monoclonal antibody to $\alpha$-tubulin and detected using a fluorescent labeled goat anti-mouse secondary antibody. DNA was stained with propidium iodide. The image of microtubules and chromatin was captured using laser scanning confocal microscope. In germinal vesicle stage oocyte, sperm chromatin remained condensation and sperm derived microtubules were not observed at 8 to 12 h after sperm injection. At 24 h after injection, the sperm nucleus developed to the metaphase chromatin along the metaphase structure of female nucleus. In some metaphase I stage oocytes, sperm chromatin decondensed at 8 h to 12 h after injection, sperm aster was seen soon after sperm injection. At 24 h after sperm injection into metaphase I stage oocyte, male chromatin developed to the metaphase chromatin while female chromatin extruded first polar body and formed the metaphase chromatin. At 12 to 15 h after sperm injection into preactivated oocytes, condensed sperm nucleus was located in close proximity of female pronucleus. However, the condensed nucleus did not fuse with female pronucleus. In preactivated ocytes, injected sperm remained condensation, a few sperm organized small microtubular aster. Instead, maternal derived microtubules were organized near the female chromatin, which seem to move condensed male chromatin near to the female pronucleus. These results suggest that sperm nuclear decondensing activity and nucleation activity of centrosome during fertilization are cell cycle dependent. In absence of male functional centrosome, female origin centrosome takes over the role of microtubule nucleation for nuclear movement.

• Journal of the Korean Society of Tobacco Science
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• v.18 no.1
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• pp.30-38
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• 1996

Meiotic metaphase chromosomes of the testis of two storage insects, tobacco moth (Ephestia elutella H.) and cigarette beetle (Lasioderma serricorne F.) were examined to study their karyotypes. The number of haploid chromosomes of the tobacco moth was 31 and the karyotype was characterized by 29 metacentric and 2 submetacentric chromosomes. The metaphase chromosome length of tobacco moth ranged ca. 1.44-2.11${\mu}{\textrm}{m}$ and the average length was 1, 78${\mu}{\textrm}{m}$. The number of haploid chromosomes of the cigarette beetle at metaphase was 10 and the karyotype was characterized by 8 metacentric and 2 submetacentric chromosomes. The range of metaphase chromosome length of the cigarette beetle was ca. 1.79- 2.39${\mu}{\textrm}{m}$ and the average length 2.09${\mu}{\textrm}{m}$.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.193-197
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• 2013

In order to investigate the effects of antioxidants on the culture of mouse preantral follicles in vitro, we examined the effects of taurine, glutathione and catalase on their growth and maturation. Addition of taurine was not effective on the survival of preantral follicles. However, metaphase II rates of oocytes within preantral follicles were significantly higher in 1 mM treated group than in control and 10 mM treated group (p<0.05). Glutathione did not improved the rates of survival and metaphase II oocytes. However, metaphase II rates of oocytes progressively decreased with increasing glutathione concentration. Catalase also showed that the rates of survival and metaphase II oocytes progressively decreased with increasing concentration. Especially, all of preantral follicles cultured in medium containing 100 IU/ml catalase were degenerated. These results suggest that low concentraion of taurine, as an antioxidant, have positive effect on the culture of mouse preantral follicles in vitro.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.11 no.6
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• pp.2124-2128
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• 2010

In this study, we determined effects of the mitogen-activated protein kinase (MAPK) inhibitor, U0126 on meiotic maturation, microtubule organization and actin filament assembly in the porcine oocyte. The phosphorylated MAPK was first detected at 12 h after the initiation of maturation cultures, fully activated at 24h, and remained until metaphase II. Treatment of germinal vesicle (GV) stage oocytes with $20{\mu}M$ U0126 completely blocked MAPK phosphorylation, but germinal vesicle breakdown (GVBD) was normally proceeded. However, the oocytes didn‘t progress to the metaphase I. The inhibition of MAPK resulted in abnormal spindles. In oocytes treated with U0126 after GVBD, polar body extrusion was normal, but the organization of the metaphase plate and chromosome segregation were abnormal. In conclusion, MAPK activity plays an important regulatory role in GV chromatin configuration and meiotic progress in porcine oocyte maturation.