• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.35-41
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• 1998

This study was carried out to investigate the development rates of human embryos co-cultured with cumulus cells to each blastocyst stage. Human zygotes were co-cultured on cumulus cell monolayer in YS medium supplemented with 20% hFF. On day 2, if patient had four or more "good" embryos (regular blastomeres without fragmentation), embryos were further cultured for 72hrs. Blastocysts on day 5 were classified into early blastocyst (ErB), early expanding blastocyst (EEB), middle expanding Blastocyst (MEB), and expanded blastocyst (EdB) on the basis of their morphological aspects of trophectoderm cells and blastocoele. Subsequently, maximum 3 of best blastocysts were transferred in 486 cycles. The results in this study were as follows: Patients who had four or more "good" embryos on day 2 were 498 persons, but patients whose embryos could not be transferred due to failure in development to the blastocyst stage on day 5 were 12 persons (2.4%). The development rate of embryos to the blastocyst stage was 58.2% (2,885/4,957) on day 5, and the rates that developed to the ErB, EEB, MEB, and EdB stage were 15.0% (743/4,957), 14.9% (739/4,957), 14.4% (714/ 4,957), and 13.9% (689/4,957), respectively. Total 1366 blastocysts were transferred in 486 cycles (mean number=2.81). The implantation rate and the ongoing implantation rate obtained by observing the number of G-sac and FHB were 29.9% (409/1,366) and 22.5% (308/1,366), respectively. The clinical pregnancy rate was 51.2% (249/486), and the ongoing pregnancy rate' was 39.1% (190/486). Among women showing ongoing pregnancy, women with singleton were 50% (95/190), women with twin were 37.9% (72/190), and women with triplet were 12.1% (23/190). Although triplet pregnancy rate in this study was high such as 12.1%, because many blastocysts with high viability were produced in our co-culture system using cumulus cells on day 5, we really believe that a multiple pregnancy except twin should not occur by selecting good embryos for maximum two blastocyst transfer. These results demonstrate that autologous cumulus cells may be used for the production of blastocysts with high developmental competence, and the use of autologous cumulus cells to be collected easily, and to be treated conveniently at OPU must be an effective means for obtaining high implantation and pregnancy rate.

• Radiation Oncology Journal
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• v.12 no.1
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• pp.27-31
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• 1994

There are a number of reports suggesting that there may be a correlation between the clinical response to radiotherapy in various tumors and the clonogenic survival of cell lines derived from these tumors following exposure to 2 Gy(SF2). Authors conducted this study to determine SF2 for cells in primary culture from surgical specimens. The tumor tissues with squamous cell carcinoma of uterine cervix and head and neck were obtained. The tumor tissues were disaggregated to single cells by incubating with collagenase type w for 2 hours with constant stirring. Single cell suspensions were inoculated in four 24-well plates precoated with cell adhesive matrix. After 24 hours of incubation at 37$ ^{\circ}C $, rows of four wells were then irradiated, consisting of control set and five other sets each receiving doses of 1,2,3,4, and 6 Gy. After incubation for a total of 13 days, the cultures were stained with crystal violet and survival at each dose was determined by quantitative image analysis system, To determine whether cell growth was of epithelial origin, immunocytochemical staining with a mixture of cytokeratin and epithelial monoclonal antibodies were performed on cell cultures. During the period of this study, we received 5 squamous cell carcinoma specimens of head and neck and 20 of uterine cervical carcinoma. Of these, 15 yielded enough cells for radiosensitivity testing. This resulted an overall success rate of 60$ \% $. The mean SF2 value for 15 tumours was 0.55$\pm$0.17 ranging from 0.20 to 0.79. These results indicate that there is a broad range of sensitivities to radiation in same histologic type. So with a large patient population, we plan to determine whether a different SF2 value is associated with tumours that are controlled with radiotherapy than those that are not.

• Physical Therapy Korea
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• v.12 no.3
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• pp.11-21
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• 2005

The present study was aimed at investigating the effect of swimming training on brain function after focal cerebral ischemia in rats. Therefore, this study was examined on neurogenesis in dentate gyrus of hippocampus using 5-bromo-2'-deoxyuridine (BrdU) to label proliferating cells and assessed the neurological response following focal cerebral ischemia in rats using neurological motor behavioral test. In an observer-blinded fashion, twenty male Sprague-Dawley (280~310 g, 7 weeks old) rats were divided into four groups: MCAO plus swimming group (ME, $n_1$=5), MCAO plus control group (MC, $n_2$=5), SHAM plus swimming group (SE, $n_3$=5), SHAM plus control group (SC, $n_4$=5). The results of this study were as follows: 1) The limb placing time before and after swimming in the ME group were significantly longer than the MC group (p<.05), the SE group were significantly longer than the SC group (p<.01). 2) The balance beam scores before and after swimming in the ME group was higher than the SE group, the MC group was higher than the SC group but was not significantly different (p>.001). 3) The foot fault index before and after swimming training in ME group was significantly lower (i.e., improved) than the MC group (p<.001) and the SE group (p<.001), the SE group was significantly lower (i.e., improved) than the SC group (p<.001). 4) The mean number of BrdU-positive cells in the dentate gyrus in the ME group was significantly higher than the MC group (p<.001) and the SE group (p<.01). The MC group and the SE group was significantly higher than the SC group (p<.001). 5) There was significantly correlation between limb placing time and number of BrdU-positive cells on swimming training, there was positive correlation (r=.807, p<.0001) and between foot fault index and BrdU-positive cells number, there was negative correlation (r=-.503, p<.05). However, between balance beam scores and BrdU-positive cells number, there was no correlation. In conclusion, the present study demonstrates that the role of swimming training improves behavioral motor function probably by enhancing cell proliferation in that hippocampus. This study provides a model for investigating the stroke rehabilitation that underlies neurogenesis and functional ability.

• Journal of Animal Science and Technology
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• v.45 no.5
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• pp.695-702
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• 2003

Nucleolus Organizer Regions (NORs) are the specific chromosome sites where ribosomal genes are located and highly expressed. We have applied the AgNOR staining to identify the distribution of NORs in the chromosomes of Korean Cattle. We have also studied the NORs pattern on the cells originated from different breeds, tissues and sex. Peripheral blood from forty-four Korean Cattle and Holstein was cultured for chromosme preparation. The fibroblast culture from biopsied ear skins was also conducted for chromosome analysis. The distribution of NORs was analyzed by sequential Ag staining and G-banding on metaphases of the cells. In Korean Cattle, the NORs are localized on the telomeres of the five chromosome pairs number 2, 3, 4, 11 and 28. The number of NORs per metaphase ranged from 2 to 10 giving a mean value of 5.6. The number of NORs per cell varied among individuals and cells within same individual. The size of NORs also differed in NO-chromosomes. The number of NORs was significantly different between Korean Cattle and Holstein, fibroblasts and lymphocytes, and male and female. However, the distribution and frequency of NORs were similar among the cells regardless of breeds, tissues, and sex.

• Korean Journal of Veterinary Research
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• v.15 no.2
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• pp.161-176
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• 1975

In order to evaluate the epizootiological influence of high-land on cattle feeding, routine hematological observation, with additional examinations of feces and general clinical condition, was conducted comparing the high-land cows resident at altitude of 800 to 1,200 meters in Daegwanryeong to the low-land cows resident at altitudes of less than 100 meters in Samcheok and Gangneung. The high-land cows were divided into 3 groups such as group A, B and C, consisting of 15, 16 and 20 adult cows respectively, and among the 3 groups only group C was exposed to abrupt high-land cold before observation. The low-land cows, that is group D, were consisted of 25 adult cows. The results obtained in the survey were summarized as follows: 1. Number of erythrocytes, concentration of hemoglobin and packed cell volume were remarkably higher (p<0.01) in all of 3 groups (group A, B and C) of high-land cows than trios of low-land cows (group D). These higher values in high-land cows were ascribable to the better feeding and hygienic management, and lesser infestation of small-type Piroplasma (Theileria) and internal parasites in contrast with the low-land cows. 2. Remarkably higher value of mean corpuscular volume (p<0.01) and a tendency to lower value of mean corpuscular hemoglobin concentration were noted in group D in contrast with group A, B and C. The reason was attributed to the higher infestation (p<0.01) of smalltype piroplasma in low-land cows in contrast with high-land cows. 3. It was noticiable that even though lesser infestation of small type Piroplama in the highland cows was shown, a natural case of clinical small-type-piroplasmosis was found in high-land group B as well as a case in the low-land group D. The blood Pattern showed characteristic macrocytic-hypochromic-anemia with increased reticulocytes in both two cases (Table 7). 4. In the observation of leukocytic series, marked increase of eosinophils in group D was seen in accordance with heavy infestation of gastrointestinal nematodes, and increased neutrophils with higher appearance of nonlobulated form and eosinopenia in group C only was observed suggesting the leukocytic response to the cold exposure in high-land. 5. Mean values of thrombocytes in group A,B,C and D were 48.4, 40.7, 42.7 and $32.3{\times}10^4/mm^3$ respcetively, and no statistically significant differences were observed.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.293-302
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• 1997

Follicular oocytes of Grade I and II were collected from 2~6 mm ovarian follicles and matured in vitro (IVM) for 24 hrs in TCM-199 su, pp.emented with 35$\mu\textrm{g}$/ml FSH, 10$\mu\textrm{g}$/ml LH, and 1$\mu\textrm{g}$/ml estradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. They were fretilized in vitro (IVF) by epididymal spermatozoa capacitated with heparin for 12 hrs. The zygotes were then co-cultured in vitro with bovine oviducted epithelial cells (BOEC) for 7 to 9 days. The optimal time for IVM, the successful enucleation of IVM oocytes by micromanipulation at different oocyte ages after IVM, and the ideal culture system for IVM for effective IVF and in vitro development of IVM-IVF embryos was examined for in vitro production of nuclear recipient oocytes and nuclear donor embryos. To improve the efficiency of nuclear transplantation (NT) of IVF embryo into IVM follicular oocytes, this study evaluated the optimal electric condition and oocytes age for activation of IVM oocytes and in vitro development of NT embryos. In vitro development of NT embryos with preactivation or non-preactivation in enucleation oocytes, cell number of IVN-IVF embryos, and NT embryos wre also examined. The results obtained were as follows; 1. The most suitable enucleation time was at 24 hpm (83.3%) rather than that of 28 hpm(69.6%) and 32 hpm(50.0%). 2. There was no difference among the fusion rates of NT embryos at the voltages of 0.75, 1.0 and 1.5 kV/cm, but the in vitro development rates to morule and blastocyst were significantly (P<0.05) higher at the voltage of 0.75(12.5%) and 1.0kV/cm (12.6%) compared to 1.5kV/cm(0%). 3. No significant difference in activation rates were seen in NT embryos stimulated for 30, 60 and 120 $\mu$sec (71.7, 85.2 and 71.9%, respectively), but the in vitro development rates to morulae and blastocyst were significantly (P<0.05) higher in the oocytes stimulated for 30 $\mu$sec (11.6%) and 60 $\mu$sec(10.7%) than 120 $\mu$sec(0.0%). 4. The fusion rates (71.0 and 87.3%) and the in vitro development rates (9.1 and 12.7%) to morula and blastocyst were seen in the NT embryos stimulated at 28 and 32 hpm under the condition of 1.0 kV/ml, 60 $\mu$sec. However, at 24 hpm the fusion rates were 64.8% and the in vitro development to morula and blastocyst were not seen. 5. The fusion rates between the 8~12, 13~17 and 18~22-cell stage of IVM-IVF embryos were not significantly different. The in vitro development rates of the fused embryos to morula and blastocyst which were received from a blastomere of 8~12, 13~17 and 18~22-cell stages of IVM-IVF embryos were 14.9, 8.3 and 6.5%, respectively. 6. The in vitro development rate of the enucleated recipient oocytes with preactivation (24.2%) to morula and blastocyst was significantly (P<0.05) higher than that of non-preactivation (12.8%). 7. The cell numbers of NT blastocyst and IVM-IVF blastocyst cultured during 7~9 days were 63$\pm$11 and 119$\pm$23, and then their the mean cell cycle number were 5.98 and 6.89, respectively.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.213-220
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• 2009

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ${\mu}g/ml$) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or $20{\sim}80{\mu}g/ml$ FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes ($85{\sim}89%$) was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage ($86{\sim}94%$) and mean number of cells in blastocyst ($33{\sim}37$ cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 ${\mu}g/ml$ FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 ${\mu}g/ml$ FSH,i numectivelo. In SCNT, fusion ($78{\sim}83%$) of cell-cytoplast couplets and siosequent embryo cleavage ($82{\sim}88%$) were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 ${\mu}g/ml$ FSHr(25% vs. $11{\sim}18%$). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 ${\mu}g/ml$ FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.261-267
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• 2012

The technique of SCNT is now well established but still remains inefficient. The in vitro development of SCNT embryos is dependent upon numerous factors including the recipient cytoplast and karyoplast. Above all, the metaphase of the second meiotic division (MII) oocytes have typically become the recipient of choice. Generally high level of MPF present in MII oocytes induces the transferred nucleus to enter mitotic division precociously and causes NEBD and PCC, which may be the critical role for nuclear reprogramming. In the present study we investigated the in vitro development and pregnancy of White-Hanwoo SCNT embryos treated with caffeine (a protein kinase phosphatase inhibitor). As results, the treatment of 10 mM caffeine for 6 h significantly increased MPF activity in bovine oocytes but does not affect the developmental competence to the blastocyst stage in bovine SCNT embryos. However, a significant increase in the mean cell number of blastocysts and the frequency of pregnant on 150 days of White-Hanwoo SCNT embryos produced using caffeine treated cytoplasts was observed. These results indicated that the recipient cytoplast treated with caffeine for a short period prior to reconstruction of SCNT embryos is able to increase the frequency of pregnancy in cow.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.2
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• pp.455-462
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• 2004

Prostate cancer is a leading cause of cancer death in American men and evidences point to significant life style/diet components as risk factors for its development or prevention. Two large cohort studies have identified the consumption of tomatoes or high Plasma levels of Iycopene as associated with reduced risk. A number of other substances such as quercetin, phytoene, phytofluene, cyclolycopene, salicylates and tomatine in tomato besides lycopene could have anticancer activity and may be acting synergistically with lycopene. Lycopene at almost physiologically feasible concentrations, reduces cell viability by cell cycle arrest and apoptosis and modulates the cyclin pathways as well as increasing intercellular communication. However, it is not clear whether lycopene or its oxidation products are more bioactive. Tomato product supplementation results in plasma accumulation of phytoene, Phytofluene, the lycopene oxidation product, and cyclolycopene at significant concentrations and lycopene supplementation, either as a tomato product or as beadlets, results in maximal mean plasma lycopene concentrations of ∼ 1 $\mu$M which is at the lower limit of its activity in cell culture. Rats and mice are poor accumulators of lycopene and other carotenoids making them poor models for the study of cancer prevention and control. Of the 19 animal studies for various cancer sites, lycopene showed a positive effect in 10 studies but negative in 2 prostate cancer studies. In vivo prevention of leukocyte DNA damage in humans has been mostly studied using tomato product supplementation but lycopene supplementation appeared to reduce oxidative DNA damage as well as tomato product supplementation. Lycopene appears to be bioactive in intefering with carcinogenesis but the actions of phytoene, phytofluene or cyclolycopene cannot be ruled out since these compounds were present in most of the lycopene material used for these studies. Although lycopene remains as a promising agent, especially for cancer control, exploring interactions with other tomato phytochemicals and with current prostate cancer therapies should be encouraged.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.65-72
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• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.