• Journal of Embryo Transfer
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• v.24 no.1
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• pp.57-64
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• 2009

This study was carried out to investigate expression of apoptosis-related differentially expressed gene (DEG) in ovaries of Korean cattle with follicular and luteal cysts and to identify the relationship between cyst and apoptosis using microarray, real-time PCR, TUNEL staining, and Western blot analysis. Microarray data showed that PIK3R2 and AKT1 were significantly up-regulated in follicular cyst, and TNF-RAF2, PRLR, FOXL2, STK4, and COL4A3 were up-regulated whereas INHA, CIDEB, BCL10, and FASLG were down-regulated in luteal cyst. Real-time PCR was performed to validate DEGs altered in luteal cyst. Of nine DEGs, four DEGs down-regulated in luteal cyst showed a positive corelation between microarray data and real-time PCR data. In this study, we focused on INHA, among many DEGs, which was highly down-regulated in both follicular and luteal cysts. Real-time PCR and micro array data showed that INHA was down-regulated by 12.3-fold and by 1.4-fold, respectively, in the bovine follicular cyst. TUNEL assay and Western blot analysis for ERK, JNK, p38, PI3K, and Akt, which were used to detect whether apoptosis is occurred, showed no significant changes in cystic ovaries (p>0.05). In the expression and activity of caspase-3, Bax, Bel-2, and Bel-xL, there was no significant changes between follicular cystic ovary and normal ovary. Rather, the expression levels of PI3K and p-Akt were decreased in follicular cystic ovary. These results suggest that deficiency of apoptosis in cystic ovary is associated with decreased expression of apoptotic effectors.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.31 no.2
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• pp.129-142
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• 1986

Fifty-five local collections of buck wheat, Fagopyrum esculentum, were investigated their ratios of long-styled (LS) and short-styled (SS) flowers, fertility, meiosis of megaspore and microspore mother cell, female and male gametogenesis, and egg apparatus in accordance with the sowing seasons (spring, summer), altitudes (20m, 50-100m, 300m), and parent style types (L, S). Also they were embryologically investigated the fertility, fertilizing phenomenon and proembryogenesis by the legitimate and illegitimate pollination. There were no differences in the ratios of long-styled and short-5tyled flowers along with altitudes, but more irregularness was observed in plain area than that in the mountaineous or coastal area. LS versus SS ratios by sowing seasons were significantly separated into 1 : 1 in the summer sowing (P 0.1), but they were irregularly separated in the spring sowing. The segregating ratios by parent style types showed more number of short-styled flower in the spring sowing, and were statistically seperated into 1 : 1 in the summer sowing (P 0.25), regardless to parent style types. In the artificial legitimate union, the seed setting rates of the summer sowing (59-61%) were much higher than those of the spring sowing (about 30%), but in the artificial illegitimate union the seed setting rates were only fructified about 0.8-1.8% in the spring sowing. The seed setting rates in accordance with flowering stages were larger in turn early, middle, late, in the summer sowing. The grain number and grain weight per plant of short-styled flower were more than those of long-styled one regardless to style types. The 1,000 grain weight of long-styled flower was heavier than that of short-styled one in large grain, but it was lighter than that of short-styled flower in small or medium grain. The percentage of normal female and male gametogenesis in the summer sowing were higher than those in the spring sowing. The ovule was atropous and two polar nuclei were a synkarion before flowering. The pollens germinated at 30 minuts after pollination and the pollen tube grew continually and penetrated into micropyle at 1.5-2 hours and the two male nuclei fertilized with egg nucleus at 3 -5 hours after pollination. Flertilizing times in summer were shorter than in autumn. The fertilized egg was divided in a small apical cell toward the interior of the embryo sac and a large basal cell toward the micropyle cell at 15-24 hours after pollination, and division times in summer were shorter than in autumn. The proembryo began the embryogenesis at 7-8 days and formed itself into the perfect embryo at 15 days after pollination.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.26 no.1
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• pp.110-114
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• 1981

These experiments were carried out to define the effects of NAA, 2, 4-D and Benzyladenine on the callus induction and the organ differentiation from the explants and to find out the vegetative propagation method of Korean ginseng. The results obtained are summarized as follows; 1. NAA was significantly effective in forming roots from the ginseng stem segment and the number of roots was increased by increasing NAA concentration in the medium. The roots were formed from both distal and proximal ends of the ginseng stem segments grown on the medium containing more than 2mg/L of NAA. 2. The amount of callus growth increased proportionatly with NAA concentration in the range of 4.0mg per liter in the medium. The callus was easly induced from stem segment than leaf segment and 2, 4-D was more effective in callus induction and growth than NAA. 3. The benzyladenine showed the significant inhibition effect in forming roots from ginseng explant. The callus was not induced with BA alone, but in BA and 2, 4-D or BA and NAA added medium, the callus was easily induced and its growth was also accelerated. The interaction effects between 2, 4-D and BA on the callus induction and growth were significantly higher than those between NAA and BA. 4. As the ginseng embryos were cultured on the M.S. medium supplemented with 2mg per liter NAA, number of shoots was significantly increased and the percentage of embryo which had shown more than 4 shoots later was 22.2%. On the medium containing 8mg per liter NAA, the ginseng embryo showed the normal growth of shoots and leaves, but increased roots and callus induction on the basal part of shoots. 5. When the shoots with 3 leaflets were cut in 1.5cm long and grown on the Blayde's medium containing NAA 1.0mg per liter, roots were formed at the proximal end of shoot, and a new ginseng seedling was successfully obtained.

• Journal of Acupuncture Research
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• v.17 no.2
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• pp.187-208
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• 2000

By the activation of ovary hormone, many morphological changes occur in the epithelial cell lines and muscle cells in rat uterus. These two cells in uterus are important to the implantation of embryo, maintaining pregnancy and starting parturition. One important change associated with the morphological change of these two cells in uterus is the change on prostaglandin(PG) metabolism. Its presence and synthesis in endometriurn and myometrium in uterus affects estrous cycle and the start of embryo implantation in uterus. It also performs as an important modulator in parturition. So the abnormally weak expression of PG causes difficulty during labor and over-expression causes pre-term labor. PG biosynthesis starts from either free or liberated arachidonic acids from membrane phospholipid by phospholipase. Such arachidonic acids are converted into PG catalyzed by Cyclooxygenase. Under normal physiological condition, Cyclooxygenase-1(COX-1) having 602 units of amino acids controls the synthesis of PG. It acts as a local hormone regulating vasomodulation of blood flow, flexible muscle movement, increasing the blood permeability and contributing the protective role in preserving integrity of the stomach lining and Cyclooxygenase-2 (COX-2) is induced by the inflammation, pregnancy and increased its expression until parturition. Lipid metabolite like PG is located in uterine and expression of COX-2 increased with pregnancy. Increased expression of COX proteins in epithelial cells and myometrial cells are told to increase the muscle contractility in uterus but decreased right after the labor in rat. It is a good sign indicating that COX proteins are deeply related to the start of labor. Currently, Several studies report the use of PG and COX-2 inhibitor as medication for controlled abortion or to prevent pre-term labor but they entail various side-effects. Our study proposed to suggest use of acupuncture as an another mediator to control abortion or pre-term labor without causing unnecessary side-effects by those medicines. Two acupuncture sites, LI-4 & SP-6 were selected due to their known efficacy. From the immunohistochemical staining of COX-2, normal expression of COX-2 protein in nonpregnant SD rat's uterus revealed that COX-2 protein was primarily detected in the lumina epithelial lining and in the epithelial cell lining contacting the stromal cells. High resolution optical microscopic scanning revealed distinguishable staining in the myometrial mucosa. LI-4 acupuncture administered nonpregnant rat's uterus showed strong expression for COX-2 in endometrium contacted with lumina epithelial lining of rat uterus and in myometrial mucosa. Stromal cells showed more staining than untreated nonpregnant rat's uterus and stronger staining in stromal cells contacting myometrial layer compared to untreated nonpregnant rat's uterus. SP-6 acupuncture administered nonpregnant rat's uterus showed weak expression for COX-2 in myometrial layers and stromal cells but no staining was visible in lumina epitheliai and glandular epithelial cells. Few stromal cells and myometrial mucosa were positively stained for COX-2. Pregnant SD rat's uterus was also immunostained for COX-2 expression after 18 days of pregnancy. Unlike to untreated nonpregnant rat's uterus, luminal epithelial cells were not positively stained for COX-2 but stronger staining for COX-2 was revealed in stromal cells. LI-4 acupunctured SD rat's uterus had very strong expression of COX-2 in luminal epithelial lining. Few stromal cells showed stronger positive COX-2 staining and myometrial layers also showed more expression than untreated pregnant rat. SP-6 acupuncture administered pregnant SD rat's uterus showed positive expression of COX-2 in epithelial cells of luminal mucosa layer but weaker than that of LI-4 acupuncture treatment's case. However, strong positive staining was revealed in stromal mucosa and myometrial layers. Virgin SD rat's uterus motility index during LI-4 acupuncture was 66.52 % (Prob〉T = 0.0197) compared to its motility before the acupuncture treatment but the motility index was slighdy elevated up to 79.58 % (Prob〉T = 0.1175) after the acupuncture. During the SP-6 acupuncture treatment for 30 minutes, uterus motility index was 90.52 % (Prob〉T = 0.1832) showing lesser decrement but consequently reached similar motility index decreasal to 79.95 % (Prob〉T = 0.0215) after the acupuncture treatment as LI-4 showed. LI-4 acupuncture tend to be a quick treatment to reducing the uterus motility in a virgin rat but eventually both two acupuncture administration created very similar reduction of uterus motility seeing the index after the both acupunctures. The uterus movement monitored during the LI-4 acupuncture administered for 30 minutes, Pregnant SD rat showed decreased motility down to 77.90 % (Prob〉 T = 0.0076) compared to uterus motility before the acupuncture and it continuously decreased down to 71.81 %(Prob〉T = 0.0214) after the removal of needle. The statistical analysis using paired t-test showed significance difference for both two motility indexs at =0.05. SP-6 acupuncture administered to pregnant SD rat also had similar pattern of decreasing uterus motility index down to 74.70 % (Prob〉T = 0.1730) during the initial 30 minutes acupuncture administration and it was continuously lowered to 71.52 % (Prob〉T = 0.0155) after the acupuncture. The paired t-test resuit for SP-6 suggest prompt response of uterus motility index to the SP-6 acupuncture treatment but consequently reached same level of inducing the motility reduction as LI-4 at =0.05 level.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.59 no.4
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• pp.398-405
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• 2014

We assessed the GABA accumulation and other components after the 'Nunkeunhukchal (BGE)', 'Josanghukchal (BR)', and 'Ilmibyeo (IB)' grain was soaked in water for 24, 36, 48, 60, 72 and 96 hr. The results showed a continuous accumulation of GABA in soaking treated brown rice of BGE and IB. Among the treated hours, 72 hours of soaking had the maximal accumulation of GABA (51.4 mg/100 g), amino acid, polyphenol and other components. The activities of glutamate decarboxylase (GAD) in brown rice and rice-bran were the same in BGE rice. However, the formation of GABA treated with L-glutamate as substrate showed dramatic increase of 354.6 (fourteen times higher than normal extraction) and 726.4 mg/100 g in BGE rice and rice-bran, respectively. These results suggested that the soaking and extraction with L-glutamate buffer could be better methods for the harvest of increased GABA.

• Korean Journal of Medicinal Crop Science
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• v.2 no.3
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• pp.219-226
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• 1994

Immature ovule of intergeneric $F_1$ hybrid between Platycodon grandiflorum x Codonopsis lanceolata for producing embryogenic callus. somatic embryos and plant regeneration were cultured in vitro on various medium as well as MT(Murashige Tucker)medium treated with different concentration of plant growth regulators. Embryogenic callus induction was highest in the treatment of NAA 0.5 $mg/{\ell}$ and zeatin 0.01 $mg/{\ell}$ added on MT medium, whereas it was lower in treatments with auxins alone. MT medium were more effective in production of somatic embryos from incubated embryogenic callus. Most favorable plant growgh regulator for producing somatic embryos was 2. 4-D 0.5 $mg/{\ell}$ and zeation. BAP 0.01$mg/{\ell}$, but hormone-free and auxins alone were less effective. NAA 0.01$mg/{\ell}$ added with zeation 0.5 $mg/{\ell}$ was effective as high as NAA 0.01 $mg/{\ell}$ alone in normal plant regeneration from somatic embryo.

• Applied Biological Chemistry
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• v.47 no.1
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• pp.61-66
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• 2004

70% ethanolic extracts were prepared from the three mutant rice cultivars with giant embryo termed Shinsunchal-giant embryonic rice, Whachung-giant embryonic rice and Nampung-giant embryonic rice, and its antioxidative and antimutagenic properties were evaluated and compared. For analysing antioxidativity, various antioxidative indices, such as electron donating ability to DPPH radical, scavenging capacity to hydroxyl radicals generated by Fenton reaction, scavenging capacity to superoxide radicals generated by HPX/XOD system, inhibitory effect on autoxidation of linoleic acid and inhibitory effect on membrane lipid peroxidation derived from rabbit erythrocyte ghost, were determined. For analysing antimutagenicity, suppressive effects on mutagenesis induced by the chemical mutagen, mitomycin C, were measured using E. coli PQ 37 as a indicator cell. The results showed that for both antioxidativity and antimutagenicity the giant embryonic rices were more effective compared to the general cooking rice, Among the giant embryonic rice cultivars, Nampung-giant embryonic rice tended to be most effective, showing its scavenging activity to DPPH radical, superoxide radical and hydroxyl radical, and inhibitory activity to lipid peroxidation was 2,3-, 3,3-, 1.7-, and 2.5-fold greater than those of normal rice, respectively.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2001.10a
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• pp.37-43
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• 2001

1. About fifty thousand of cattle embryos were transferred and 16000 ET-calves were born in 1999. Eighty percents of embryos were collected from Japanese Black beef donors and transferred to dairy Holstein heifers and cows. Since 1985, we have achieved in bovine in vitro fertilization using immature oocytes collected from ovaries of slaughterhouse. Now over 8000 embryos fertilized by Japanese Black bull, as Kitaguni 7~8 or Mitsufuku, famousbulls as high marbling score of progeny tests were sold to dairy farmers and transferred to their dairy cattle every year. 2. Embryo splitting for identical twins is demonstrated an useful tool to supply a bull for semen collection and a steer for beef performance test. According to the data of Dr. Hashiyada(2001), 296 pairs of split-half embryos were transferred to recipients and 98 gave births of 112 calves (23 pairs of identical twins and 66 singletons). 3. A blastomere-nuclear-transferred cloned calf was born in 1990 by a joint research with Drs. Tsunoda, National Institute of Animal Industry (NIAI) and Ushijima, Chiba Prefectural Farm Animal Center. The fruits of this technology were applied to the production of a calf from a cell of long-term-cultured inner cell mass (1988, Itoh et al, ZEN-NOH Central Research Institute for Feed and Livestock) and a cloned calf from three-successive-cloning (1997, Tsunoda et al.). According to the survey of MAFF of Japan, over 500 calves were born until this year and a glaf of them were already brought to the market for beef. 4. After the report of "Dolly", in February 1997, the first somatic cell clone female calves were born in July 1998 as the fruits of the joint research organized by Dr. Tsunoda in Kinki University (Kato et al, 2000). The male calves were born in August and September 1998 by the collaboration with NIAI and Kagoshima Prefecture. Then 244 calves, four pigs and a kid of goat were now born in 36 institutes of Japan. 5. Somatic cell cloning in farm animal production will bring us as effective reproductive method of elite-dairy- cows, super-cows and excellent bulls. The effect of making copy farm animal is also related to the reservation of genetic resources and re-creation of a male bull from a castrated steer of excellent marbling beef. Cloning of genetically modified animals is most promising to making pig organs transplant to people and providing protein drugs in milk of pig, goat and cattle. 6. Farm animal cloning is one of the most dreamful technologies of 21th century. It is necessary to develop this technology more efficient and stable as realistic technology of the farm animal production. We are making researches related to the best condition of donor cells for high productivity of cloning, genetic analysis of cloned animals, growth and performance abilities of clone cattle and pathological and genetical analysis of high rates of abortion and stillbirth of clone calves (about 30% of periparutum mortality). 7. It is requested in the report of Ministry of Health, labor and Welfare to make clear that carbon-copy cattle(somatic cell clone cattle) are safe and heathy for a commercial market since the somatic cell cloning is a completely new technology. Fattened beef steers (well-proved normal growth) and milking cows(shown a good fertility) are now provided for the assessment of food safety.

• Journal of The Korean Society of Grassland and Forage Science
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• v.28 no.3
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• pp.171-176
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• 2008

Efficient plant regeneration system of birdsfoot trefoil (Lotus corniculatus L.) was development. The factors affecting the somatic embryo formation, its proliferation and regeneration capacity of leaf and stem explants of Empire cultivar was investigated. The highest number of somatic embryos was obtained on B5 medium supplemented with 1 mg/L NAA and 1 mg/L BA. Depending on different explants, highest frequency of embryogenic callus and regeneration were observed in Empire with leaf explants. The response from stem explants was slower and callus induction was less than that from leaf explants. Regenerated shoots formed complete plantlets in on 1/2 MS medium supplemented with 1 mg/L IBA. Regenerated plants were morphologically uniform with normal shape and growth pattern.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.22-22
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• 2001

To produce reconstituted rabbit embryos with fetal fibroblasts, the present study was evaluated the efficiencies of the different fusion and activation conditions as assessments of subsequent development and chromosome in the embryos. New Zealand White rabbits were used throughout the study. Fetal fibroblasts collected from 22-d of fetuses were cultured in DMEM ＋ 10% FBS in 5% $CO_2$ in air. The culture was maintained for 10 passages. In every passage half of cell suspension were kept In frozen. From rabbits treated with FSH in 30% PVP solution and hCG, oocytes were surgically collected from oviducts at 14 h post-hCG injection and stripped off their cumulus cells by re-pipetting in a 300 IU hyaluronidase solution. Oocytes with an extruded first polar body and dense cytoplasm were enucleated by micromanipulation in Ham's F-10 medium＋7.5 g/$m\ell$ cytochalasin B. Euncleation was confirmed under a fluorescence microscope after staining with 5 g/$m\ell$ bisbenzimide for 2 min. Each enucleated oocyte was injected with a fetal fibroblast into a perivitelline space. Reconstructed eggs were compared fusion rates either at 2.0 ㎸/cm or 1.6 ㎸/cm(60 sec, double pulses). After fusion, all eggs were activated with the combination of 5 M ionomycin (5 min) and 10 g/$m\ell$ cycloheximide (CHX, 3h), and cultured in CRlaa medium and transferred into TCM199＋10% FBS on day 3. Although there was not significantly differ in fusion rate between treatments (60%, 2.0 ㎸/cm vs. 79.4%, 1.6 ㎸/cm), none of them in the eggs fused with 2.0 ㎸/cm developed to blastocyst. In comparison of development and chromosome status between different activation treatments (Group 1; 5 M ionomycin/10 g/$m\ell$ CHX, Group 2; 5 M ionomycin/5 g/$m\ell$ CHX ＋ 2 mM DMAP after fusion with 1.6 ㎸/cm), there were not differ in cleavage and development rates (67.3% and 28.9% in Group 1; 67% and 33% in Group 2). All out of 8 embryos evaluated in Group 1 appeared a normal diploid chromosome sets and mean number of cells (Mean SEM) on day 4.5 of culture was 141.5 23.15 (n=8). It can be concluded that the use of cycloheximide has not happened in chromosome abnormalities, and fetal fibroblasts can be used for cloning in rabbit.