Search Results

OBJECTIVE: This study was to determine the effect of different concentration of calcium in medium on the preimplantational development of zygotes and early 2-cell embryos. METHODS: Female mice of ICR strain (5~8 weeks old) were superovulated and mated with fertile males. Zygotes or early 2-cell embryos were collected by flushing the oviducts 31~32 hours after hCG injection. The embryos were cultured in various concentrations of Ca2+ in medium or with EDTA, EGTA and Ni2+. RESULT AND CONCLUSION: Treatment of high concentration of Ca2+ (3.42 mM (2X)~17.1 mM (10X) in medium didn't develop well compared to the control Low concentrations of Ca2+ (0.214 mM (1/8X)~0.855 mM (1/2X)) were deterimental to development beyond 2-cell stage. EDTA, Ca2+ chelating agent was treated with ranged concentrations of eDTA (0.014 mM~0.107 mM) to medium contaning 1.71 mM Ca2+ showed beneficial effect to development to blastocyst compared to the control. EGTA, extracellular Ca2+ chelator, was treated with ranged concentrations of EGTA (0.014~0.107 mM) to the medium contaning 1.71 mM Ca2+. There is no significant difference with the control. Ni2+ (50 micrometer), T-type Ca2+-channel blocker was treated to medium contaning low concentration of Ca2+. It overcame 2-cell block significantly. Rate of degenerated embryos decreased and developmental rate to morula and blastocyst increased more than low Ca2+ concentration alone. Further studies are needed for the overcoming effect of 2-cell block by Ni2+.
Animals ; Blastocyst ; Calcium ; Edetic Acid ; Egtazic Acid ; Embryonic Structures* ; Female ; Flushing ; Humans ; Male ; Mice* ; Morula ; Oviducts ; Zygote

4.Eetection of treponema pallidum by polymerase chain reaction.

Kee Yang CHUNG ; Jung Bock LEE

Korean Journal of Dermatology 1993;31(4):481-494

BACKGROUND: Definite criteria for the diagnosis and treatment evaluation for different clinical stags of syphilis are not yet present dute to the inability to dultivate Treponema pallidum in vitro. However, as the staining methods and the serological tests currently used have their limitations, a more definite method for its confirmation is needed. Polymerase chain reaction (PCR), due to its high sensitivity and specificity, is currently being applied to the detection of T. pallidum. OBJECTIVE: We have used PCR for the detection of T. pallidum DNA in various clinical specimens in orber to evaluate its potenital as a diagnostic tool. METHOD: Clinical specimens were collected from patients with different stages of syphilis who visited ithe Deparment of Dermatology of Yonsei medical Center for 1 year beginning from May, 1991. Sera from 63 patients, cerebrospinal fluids from 24 patients, amniotic fluids from 3 patients, and 21 tissues from 19 patients were subjected to PCR and the results were analyzed to evaluate its usefulness as a diagnostic and treatment evaluation tool. A portion of the T. pallidum-specific chromosomal DNA, tpp47, which encodes the 47 kDa surface protein, was used as the template DNA to amplify the 658 bp DNA fragment, and the following results were obtained. PCR using primers 47-1 and 47-2 were applied to amplify 658 bp DNA fragments from the T. pallidum-specific tpp47 gene encoding 47 kDa surface protein. RESULT: 1. To evaluate the sensitivity of the PCR, T. pallida and their chromosomal DNA were diluted. The diluents contataining a single organism and 1 fg of the chromosomal DNA showed positive reactions by the amplification. 2. Specificity of the PCR was determined by using T. pallidum, 4 species beloging to genus Treponema, and 9 species of nonpathogenic or pathogenic organisms. A positive reaction was obtained only when T. pallidum chromosomal DNA was used. 3. PCR was positive in 5 of 9 (55%) sera in primary syphilis, 22 of 26(84%) in secondary syphilis, 3 to 15(20%) in early latent syphilis, 1 of 19 (11%0 in late latent syphilis, 2 of 2 (100%) in neurosyphlis, and 0 of 2 (0%) in congenital syphilis. The differences in the positive rates were statistically significant (P<0.01) in all stages except neurosyphilis and congenital syphilis, as their numbers were too smalll to deduce any significant meaning. Despite their high VDRL titers, the positive rate in early latent syphilis was relatively low when compared to the rate in secondary syphilis. 4. Follw-up PCR of sera in some patients showed positive results 9 months after treatment. However, some with negative PCR before treatment showed positive results after treatment. 5. PCR was positive in 1 of 1 (50%) cerebrospinal fluid in primary syphilis, 3 of 14 (21%) in secondary syphilis, 2 of 7 (29%) in early latent syphilis, and 1 of 1(100%) in neurosyphlis. The differences in the positive rates showed no statistical significance in relation to the clinical stages. Cerebrospinal fluid VDRL test, white blood cell count, and protein content showed no correlation to the PCR results in early syphilis patients. 6. Amniotic fluid showed a positive PCR result only in a pregnant woman whose serum showed a high VDRL titer and a positive PCR. 7. PCR positive rates were 90% in frozed tissues and 50% in paraffin embedded tissues. CONCLUSION: From the results, it is suggested that PCR is not suitable for treatment evaluation but is useful for the detection of T. pallidum in sera of secondary syphilis patients and syphilitic lesions, and for the confirmation of the diagnosis the diagnosis in these cases.
Amniotic Fluid ; Cerebrospinal Fluid ; Dermatology ; Diagnosis ; DNA ; Female ; Humans ; Leukocyte Count ; Neurosyphilis ; Paraffin ; Polymerase Chain Reaction* ; Pregnant Women ; Sensitivity and Specificity ; Serologic Tests ; Syphilis ; Syphilis, Congenital ; Syphilis, Latent ; Treponema pallidum* ; Treponema*

5.Vitrification and Ultrarapid Freezing of Day 2 Mouse Embryos.

Jung Sook YANG ; Cherl SOHN ; In Ha BAE

Korean Journal of Fertility and Sterility 2000;27(3):283-289

OBJECTIVE: The study was performed to compare the survival rate and the development of day 2 mouse embryos which had freezing procedures done. METHODS: We used three different vitrification solutions (EFS, VS14, DPS) and a ultrarapid freezing solution (UFS) for cryopreservation of day 2 mouse embryo. RESULTS: We tested toxicity by exposing embryos to vitrification solutions and a ultrarapid freezing solution. The survival rates are 100%, 97.8%, 95.6% and 100% (EFS, VS14, DPS and UFS). After cultured for 96 hours, hatching rates of each group are 93.5% (no freezing), 95.6% (EFS), 86.4% (VS14), 93.0% (DPS), and 93.0% (UFS). There is no significant differences among groups. The survival rates after thawing cryopreserved embryos are 80.2%, 91.7%, 69.5%, 0% and 91.8% (slow freezing, EFS, VS14, DPS and UFS). Also cultured for 96 hours, the hatching rates are 93.5% (no freezing), 84.1% (slow freezing), 93.9%) (EFS), 48.5% (VS14) and 70.1% (UFS). CONCLUSION:The survival rates of vitrification in EFS solution and ultrarapid freezing are higher than slow freezing (p<0.05). The hatching rate of vitrification in EFS solution cultured for 96 hours is highest, so vitrification of day 2 mouse embryos in EFS solution considered as more effective for cryopreservation.
Animals ; Cryopreservation ; Embryonic Structures* ; Freezing* ; Mice* ; Survival Rate ; Vitrification*