• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.317-322
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• 2002

Objective: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. Materials and methods: A total 964 blastocysts (early${\sim}$hatched) was exposed to PI (n=831) (group I: $\leq$ 10; II: $11{\sim}15$; III: $16{\sim}20$; IV: $\geq$21 sec) and bisbenzimide (n=133) (group A: $\leq$1; B: $1{\sim}$3; C: $\geq$ 4 hr) in several periods for differential staining. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values < 0.05 were accepted as statistically significant. Results: In case of PI exposure, differential staining rates were significantly higher (p<0.05) in group I (89.8%) than in any others (group II: 77.6%; III: 29.6%; IV: 22.2%) and higher (p<0.05) in group II than in group III and IV. In case of bisbenzimide exposure, differential staining rates were not statistically differences in three groups (group A: 97.4%; B: 87.8%; C: 93.3%). Conclusion: The differential staining rates of mouse blastocysts are not affected by the exposure length of bisbenzimide. However, blastocysts were exposed to PI with period of shorter than 15 sec show best outcomes of differential staining rates.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.27-34
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• 1996

The objective of this study was to examine the cell number of Total, ICM and TE cells of bovine blastocysts according to development progression cultured in CR1 medium, which was reported as successfully supporting medium for preimplantaion bovine embryo development to the blastocyst stage, by differential labelling of the nuclei with immunosurgery and polynucleot-ide-specific fluorochromes. Blastocysts were obtained at day 8 after in vitro fertilization and classified to early, middle, expanded stage according to the developmental morphology; blastocoel expansion and zona thickness. Also, bias tocysts in the same category were divided into two parts to check the Total cell number by using bisbenzimide only and ICM, TE and Total cell number by using immunosurgery and two polynucleotide-specific fluorochromes. 1) The development rate of blastocysts at day 8 after in vitro fertilization was 29.3% and classified bIas tocysts to early, middle, expanded and hatching stage were 8.7, 9.9, 7.6 and 3.1%, respectively. 2) The numbers of total blastomere using bisbenzimide in the classified blastocysts to early, middie and expanded were 46.9${\pm}$8.6, 66.2${\pm}$12.5 and 122.8 ${\pm}$ 14.4, respectively. This indicated that CR1 is a appropriate culture medium for bovine embryo development. 3) The count of ICM and TE cell number by using differential labelling with immunosurgery and polynucleotide-specific fluorochromes in the classified blastocysts to early, middle and expanded; ICM cell numbers of were 12.8${\pm}$5.9, 26.3${\pm}$8.4 and 35.5${\pm}$15.0, respectively and TE cell numbers were 30.5${\pm}$5.0, 4 41.3${\pm}$8.2 and 81.1${\pm}$13.4, respectively. These results presented that the increase of ICM and TE cell numbers averaged two and three doublings between early and expanded blastocyst stage and also total cell number counted from ICM nuclei and TE nuclei by using differential label-ling showed the increase pattern with development advance level and the results were similar to total cell number obtained from bisbenzimide treatment only. Therefore, the differential labelling of ICM and TE nuclei in situ is a very useful technique to evaluate embryo qualities and can be used as an indicator on study of preim-plantation embryo development.

• The Korean Journal of Mycology
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• v.22 no.2
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• pp.117-121
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• 1994

Five strains of Pleurotus sajor-caju were collected from some countries including India and Papua New Guinea. Four strains produced brown fruitbody but the other strain from Papua New Guinea produced white fruitbody. Monokaryons obtained from both strains producing brown fruitbody and white fruitbody were mated each other. They showed different mating types such as brown strain of $A_1A_2B_1B_2$ and white strain of $A_3A_4B_3B_4$. DNAs were isolated from mycelia of five strains of P. sajor-caju. Mitochondrial DNA was seperated from nuclear DNA by bisbenzimide-CsCl ultracentrifugation. Digestion of the fungal mitochondrial DNA with Eco RI restriction endonuclease yielded from ten to fourteen fragments. Two strains of the five strains showed different restriction pattern of mitochondrial DNA from the other three strains. Summation of the fragment sizes gave a mitochondrial DNA size of about 60 to 65kb.

• Environmental Mutagens and Carcinogens
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• v.22 no.3
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• pp.199-204
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• 2002

It has been known that Ganoderma lucidum and Lentinus edodes have anticancer activity and immune enhancing activity. These two mushrooms were grown in liquid culture and harvested. From these mycelia, DNA was isolated and EtBr-CsCl density gradient ultracentrifugation was performed to purify it further. Then mitochondrial DNA was isolated by bisbenzimide-CsCl density ultracentrifugaton. Mitochondrial DNA of Ganoderma lucidum was digested by restriction enzymes, EcoR I, Hind Ⅲ, and Pst I, then electrophoresed. It showed 12, 22, 4 fragments. Mitochondrial DNA of Lentinus edodes was digested by EcoR I. Electric pattern showed 6 fragments. 4 fragments had appeared by Pst 1 digested mitochondrial DNA. Hind ill couldn't digest mitochondrial DNA of Lentinus edodes. Mitochondrial DNA of fusants was isolated to compare to those of parents. The results showed that fusant P₂S₄has new, recombined mitochondrial DNA. But P₂S₄had the same DNA that Ganoderma lucidum had.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.83-83
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• 2002

This study was to compare the characteristics of parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Mouse oocytes were recovered from superovulated 4wks hybrid F1 (C57BL/6xCBA/N) female mice. The oocytes were treated with 7% ethanol for 5 min and 5 ㎍/㎖ cytochalasin-B for 4 h. For IVF, the oocytes were inseminated with epididymal sperm of hybrid Fl male mice (1×10/sup 6//㎖). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count in blastocysts was carried out differential labelling using propidium iodide (red) and bisbenzimide(blue). (omitted)

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.25-32
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• 1996

This work has been carried out to examine the number of Total, ICM and TE cells of F1 mouse blastcysts at day 4 after IVF by differential labelling of the nuclei with polynucleotide-specific fluorochromes and to obtain a fundamental information of preimplantation mouse embryo development. Blastocysts produced by superovulated B6CBA F1(C57BL/${\times}$CBA) eggs were inseminated with $1{\times}10^6$spermatozoa/ml and cultured in M16 medium at $37^{\circ}C$, 5% $CO_2$ incubator for 95hrs. Blastocysts were classified as early, middle, expanded and hatching stage according to the developmental morphology; blastocoel expansion and zona thickness. The results obtained in these experiments were summarized as follows; 1) The development rate of blastocysts at 95hrs after IVF was 86.7% and classified blastocysts to early, middle, expanded and hatching were 16.3%, 18.9%, 10.5% and 40.9%, respectively. 2) The numbers of total blastomere using bisbenzimide in the classified blastocysts to early, middle, expanded and hatching were 35.6${\pm}$1O.4, 49.4${\pm}$8.6, 60.8${\pm}$1O.7 and 62.7${\pm}$13.9, respectively. 3) In ICM and TE cell number by using differential labelling with polynucleotide-specific fluorochrome in the classified blastocysts to early, middle, expanded and hatching; ICM numbers were 9.6${\pm}$3.0, 13.6${\pm}$3.9, 16.0${\pm}$3.3 and 19.5${\pm}$4.6, respectively and TE cell numbers were 30.6${\pm}$5.1, 39.9${\pm}$5.8, 42.2${\pm}$8.1 and 43.7${\pm}$11.1, respectively. These results showed the same increase pattern according to development advance level. Also, when compared with the results of total count were obtained between bisbenzimide only and differential labelling, both of them showed the same increase pattern according to development level and at the same time their cell numbers were almost the same. So, rapid and simple cell count method using differential labelling can be used for the examination of later preimplantation development or as an indicator of embryo quality according to the variables of culture conditions.

• The Korean Journal of Ecology
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• v.18 no.1
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• pp.109-120
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• 1995

As the genetically modified microorganisms (GMMs) and their recombinant plasmid DNAs could be released into natural environments, their stabilities and impacts to indigenous microorganisls have become very importhant research subjects concerning with environmental and ecological aspects. In this study, the genetically modified E. coli CU103 and its recombinant pCU103 plasmid DNA, in which pcbCD genes involving in degradation of biphenyl and 4-chlorobiphenyl were cloned, were studied for their survival and stability in several different waters established under laboratory conditions. E. coli CU103 and its host E. coli XL1-Blue survived longer in sterile distilled water (SDW) and filtered autoclaved river water (FAW) than in filtered river water (FW). A lot of extracellular DNAs were released from E. coli CU103 by lytic action of phages in FW and the released DNAs were degraded by DNase dissolved in the water. Such effects of the factors in FW on stability of the recombinant pCU103 plasmid were also observed in the results of gel electrophoresis, quantitative analysis with bisbenzimide, and transformation assay. Therefore, the recombinant plasmids of pCU103 were found to be readily liberated from the genetically modified E. coli CU103 into waters by normal metabolic processes and lysis of cells. And the plasmid DNAs were quite stable in waters, but their stabilities could be affected by physicoKDICical and biological factors in non-sterile natural waters.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.303-310
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• 1996

The objective of this study was to examine the effect of embryos development following IVF of in vitro-matured porcine oocytes treated with epidermal growth factor (EGF). When cumulus-enclosed oocytes were incubated in TCM 199 medium supplemented with (1) control group, (2) 10 ng/ml EGF, (3) 10${\mu}g$ml FSH and 10% FBS, or (4) 10 ng/ml EGF, 10 ${\mu}g$/ml FSH, and 10% FBS for 42 hr, the late developmental rates on NCSU (0.4% BSA) medium after fertilization were higher in (3) and (4) groups (13.4, 18.3%) than in (2) group (5.2%, p < 0.005), but (2) group is significantly higher than the development to blastocyst of oocytes of (1) group (1.2%). Also, when the cell number of total, ICM, and TE of those blastocysts at 6 day produced in vitro was investigated by double staining (PI and bisbenzimide), total cell number of (4) group (58.80${\pm}$ 11.90) was higher than that of (2) and (3) groups (42.17${\pm}$9.97, 49.07${\pm}$9.77, P < 0.05). ICM cell number of blastocysts of (4) group (11.69${\pm}$5.56) was higher than that of (2) and (3) groups (5.00${\pm}$4.24, 6.77${\pm}$4. 92, P < 0.05). Furthermore, the proportion of ICM in (4) group (19.0${\pm}$1.6) was higher than that in (2) and (3) groups (11.1${\pm}$3.0, 12. 7${\pm}$2.1). These results suggested that in vitromatured porcine oocytes treated with EGF alone can be developed to blastocyst, but high proportion on the development to blastocyst and number of total cell and ICM in blastocyst can be obtained when supplemented with additional FSH and FBS.

• Animal cells and systems
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• v.15 no.4
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• pp.287-294
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• 2011

The accelerated cooling rate associated with vitrification reduces injuries attributed to cryopreservation and improves the post-freezing developmental competence of vitrified embryos. In this study, embryos were vitrified and warmed and morphologically evaluated for their development to blastocysts. Survival rates between the fresh ($96.7%{\pm}3.8%$) and vitrified embryos ($90.7%{\pm}5.1%$) did not differ significantly (P>0.05). The mitochondrial membrane potential of fresh control cells measured by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanide iodide staining was similar to that of cryoprotected and vitrified embryos. Mitochondrial staining with rhodamine 123 did not differ among the fresh, cryoprotected, and vitrified embryos. Moreover, the distribution of $H_2O_2$, assessed by 2',7'-dichlorodihydrofluorescein diacetate staining, did not differ among the groups. The results showed that the developmental rate did not differ significantly among the fresh ($87.8%{\pm}11.3%$), cryoprotected ($83.2%{\pm}7.6%$), and vitrified 2-cell embryos ($75.8%{\pm}14.2%$). The mean number of the inner cell mass (ICM), trophectoderm (TE), and apoptotic cells was counted and statistically compared, and although the number of ICM and TE was decreased in the cryoprotected and vitrified embryos, there were no significant differences among the groups (P>0.05). During the cultivation period, randomly selected blastocysts from each group were stained using either 4',6-diamidino-2-phenylindole and bisbenzimide or the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling technique. The incidence of apoptosis appeared to be almost identical in all the groups. Droplet vitrification could subsequently lead to high survival and developmental rates of cryopreserved mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.1-7
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• 2004

연구 목적: 본 연구의 목적은 마우스에서 배양액의 용량이 배반포 배 형성과 세포수에 미치는 영향을 조사하기 위하여 실시하였다. 연구 재료 및 방법: $3{\sim}4$주령 ICR 암마우스에게 48시간 간격으로 5 IU PMSG와 hCG 주사 후 (hCG 주사 후 수컷과 동숙) $46{\sim}50$시간에 난관으로부터 총 138개의 2-세포기 배를 회수하여 2 ml (group I) 또는 $50{\mu}l$ (group II)의 배양액 (Dulbecco's Modified Eagle Medium + 20% human follicular fluid)에서 72시간 동안 배반포기까지 배양하였다. 배반포 배는 zona-intact (ZiB)와 zona-escape (ZeB)로 등급을 구분하고 나서, propidium iodide와 bisbenzimide를 이용한 differential staining 방법으로 염색하여 평균 세포수, 내세포괴(ICM) 세포수, 영양배엽(TE) 세포수, 총 세포수에 대한 ICM의 비율 (%ICM) 및 ICM:TE 비율을 조사하였다. 결과에 대한 유의성 검정은 $X^2$ test와 t-test를 이용하였으며, p<0.05일 때 통계적인 차이가 있는 것으로 하였다. 결 과: Group I과 II에서, 총 배반포 ($62.3{\pm}20.7%$ vs. $63.8{\pm}22.9%$), ZiB ($31.9{\pm}24.0%$ vs. $30.4{\pm}18.2%$)와 ZeB 형성율 ($30.4{\pm}20.8%$ vs. $33.3{\pm}22.3%$)은 차이가 없었다. 87개의 배반포 배를 염색 시도하였는데, 명확하게 differential staining된 41개의 배반포 배만을 대상으로 세포수를 조사하였다. 평균 세포수 ($61.6{\pm}19.5$ vs. $63.7{\pm}26.8$), ICM 세포수 ($13.0{\pm}10.6$ vs. $12.8{\pm}10.5$), TE 세포수 ($49.0{\pm}19.0$ vs. $47.8{\pm}18.7$), %ICM ($21.0{\pm}12.6%$ vs. $21.1{\pm}13.2%$) 및 ICM:TE 비율 (1:$3.77{\pm}4.9$ vs. 1:$3.72{\pm}4.8$)에서도 group I과 II에서 차이가 없었다. 결 론: 마우스에서 배 발생 능력의 척도로 쓰이는 배반포 배 형성율 배반포 배의 등급 세포수 및 %ICM 등이 20% 난포액을 첨가한 MEM 배양액의 용량에 따라 영향을 받지 않았다.