• Journal of Embryo Transfer
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• v.11 no.2
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• pp.111-124
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• 1996

The present study was carried out to investigate the effects of co-culture cells and growth factors on in vitro culture of Korean native cattle(KNC) embryos fertilized in vitro. Two-eight cell embryos were cultured in vitro using 4 types of co-culture cells and 3 growth factors singly or in combination. The results were as follows, In the co-culture of 2~8 cell embryos with bovine oviductal epithelial cell(BOEC), granulosa cell(BGC), uterine epithelial cell(BUEC) and mouse embryonic fibroblast (MEF) monolayers, the developing rate to blastocysts was significantly(P<0.05) higher with BUEC(32.1%) than with MEF(15.3%), BGC(13.2%) and non co-culture control(11.6%). When the morula co-cultured with BOEC for 5 days following in vitro fertilization were co-cultured with BOEC continuously or with BUEC, respectively, the developing rate to blastocysts was higher with BUEC(73.9%) than with BOEC(56.0%). To examine the effects of growth factors on in vitro development of 2~8 cell embryos, epidermal growth factor(EGF), transforming growth factor-$\beta$l(TGF-$\beta$l) and insulin-like growth factor-1(IGF-1) were added singly or in combination to TCM 199 maturation medium with respective concentration. In a addition of each 10, 30 and SOng /rnl EGF, the developing rate to blastocysts was the highest in lOng /ml EGF(25.3%). In addition of each 1, 2 and Sng /mi TGF-$\beta$1, the developing rate to blastocysts was the highest in lng /ml TGF-$\beta$1(28.8%). In addition of each 50, 100ng/ml JGF-l, the developing rate to blastocysts was higher in 100ng/ml IGF-l(16.5%) than in SOng/mi IGF-1(12.9%). When lOng /ml EGF and lng /ml TGF-$\beta$l was added singly or in combination, the developing rate to blastocysts was similar in groups added singly or in combination with EGF and TGF-$\beta$l (23.l~24.6%), although higher than in control(16.7%). In the co-culture of 2~8 cell embryos Wth BOEC + each 10, 30 and 5Ong /rnl EGF, the developing rate to blastocysts was significantly(p<0.05) higher in BOEC + long /ml EGF(32.3%) than in BOEC + 3Ong /ml EGF(18.9%) and BOEC + song /ml EGF(9.7%). In the co-culture of 2~8 cell embryos with BOEC + each 1, 2, Sng /ml TGF-$\beta$l the developing rate to blastocysts was higher in BOEC + Sng/rnl TGF-$\beta$l(28.2%) than in BOEC + lng /ml TGF-$\beta$l(21.7%) and BOEC + 2ng/ml TGF-$\beta$l(21.4%). In summary, higher developing rate to blastocysts were obtained with co-culture of BUEC for co-culture system, with addition of lOng /ml EGF or lng /ml TGF-$\beta$l for growth factor culture system, and with co-culture of BOEC + lOng /ml EGF or BOEC + Sng /ml TGF-$\beta$l for co-culture + growth factor culture system.

• Journal of Life Science
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• v.20 no.8
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• pp.1268-1275
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• 2010

This study was carried out to investigate the anti-proliferative activity of solid-state fermented medicinal herbs which include Phellinus baumii. Methanol extracts were prepared from 36 different medicinal herbs and their fermented counterparts. These extracts were used to treat human colorectal HCT116 cell, human embryonic kidney cell HEK-293, pre-adipocyte cell 3T3-L1, and pre-osteoblast cell MC3T3-E1 for 24 hr. At a concentration of 100 ${\mu}g/ml$, the extracts of Amomum villosum, Cnidium officinale Makino, Dendrobium moniliforme, Dictamnus dasycarpus, Diospyros kaki Thunb, Eucommia ulmoides Oliv, Ginkgo biloba L, Magnolia denudata Desrousseaux, Orostachys japonicus, Panax notoginseng, Pharbitis nil Choisy, Polygala tenuifolia and Trichosanthes kirilowii (seed) led to a < 50% decrease in cell proliferation, and mycelium of P. baumii showed a 46.3% decrease in cell proliferation. Meanwhile, the extracts of the 25 fermented herbs showed similar anti-proliferative activities compared to those of individual non-fermented herbs. However, the extracts of the fermented Drynaria fortunei Kunze (1), Lycium chinense Mill (2), Fritillaria thunbergii Miquel (3) and Prunus persica showed increased anti-proliferative activity. The $IC_{50}s$ of (1), (2) and (3) were especially decreased to 28, 85 and 80 ${\mu}g/ml$ from 394, 917 and 149 ${\mu}g/ml$, respectively. Furthermore, the cytotoxicity of the extracts of fermented (1), (2) and (3) against HEK-293, 3T3-L1, and MC3T3-E1was negligible up to 200 ${\mu}g/ml$. These results suggest that solid-state fermentation using the mycellium of P. baumiiproduce potential anti-cancer agents or strengthen the bioactivity of medicinal herbs.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.1
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• pp.199-203
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• 2012

Purpose: To evaluate the prognostic value of serum CYFRA21-1, CEA and hemoglobin levels regarding long-term survival of patients with esophageal squamous cell carcinoma (ESCC) treated with concurrent chemoradiotherapy (CRT). Methods: Age, gender, Karnofsky Performance Status (KPS), tumor location, tumor length, T stage, N stage and serum hemoglobin, and CYFRA21-1 and CEA levels before concurrent CRT were retrospectively investigated and related to outcome in 113 patients receiving 5-fluorouracil and cisplatin combined with radiotherapy for ESCC. The Kaplan-Meier method was used to analyze prognosis, the log-rank to compare groups, the Cox proportional hazards model for multivariate analysis, and ROC curve analysis for assessment of predictive performance of biologic markers. Results: The median survival time was 20.1 months and the 1-, 2-, 3-, 5- year overall survival rates were 66.4%, 43.4%, 31.9% and 15.0%, respectively. Univariate analysis showed that factors associated with prognosis were KPS, tumor length, T-stage, N-stage, hemoglobin, CYFRA21-1 and CEA level. Multivariate analysis showed T-stage, N-stage, hemoglobin, CYFRA21-1 and CEA level were independent predictors of prognosis. By ROC curve, CYFRA21-1 and hemoglobin showed better predictive performance for OS than CEA (AUC= 0.791, 0.704, 0.545; P=0.000, 0.000, 0.409). Conclusions: Of all clinicopathological and molecular factors, T stage, N stage, hemoglobin, CYFRA21-1 and CEA level were independent predictors of prognosis for patients with ESCC treated with concurrent CRT. Among biomarkers, CYFRA21-1 and hemoglobin may have a better predictive potential than CEA for long-term outcomes.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.2
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• pp.90-95
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• 2017

Objective: We investigated whether the insemination method (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]) affected morphokinetic events and abnormal cleavage events in embryonic development. Methods: A total of 1,830 normal fertilized embryos were obtained from 272 IVF and ICSI cycles that underwent ovum retrieval culture using a time-lapse system (Embryoscope) from June 2013 to March 2015. All embryos were investigated by a detailed time-lapse analysis that measured the developmental events in the hours after IVF or ICSI insemination. Results: No significant differences were observed between the two groups regarding clinical outcomes (p>0.05). ICSI-derived embryos showed significantly faster morphokinetics than those derived from conventional IVF, from the time to pronuclear fading to the time to 6 cells (p<0.05). However, no significant differences were found from the time to 7 cells to the time to expanded blastocyst (p>0.05). There were no differences in abnormal cleavage events between the two groups (p>0.05); they showed the same rates of direct cleavage from 1 to 3 cells, 2 multinucleated cells, 2 uneven cells, and reverse cleavage. Conclusion: The morphokinetics of embryo development was found to vary between IVF- and ICSI-fertilized oocytes, at least until the 6-cell stage. However, these differences did not affect the clinical outcomes of the embryo. Additionally, no significant differences in abnormal cleavage events were found according to the fertilization method.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.43-48
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• 1995

The purpose of this study was to examine the suvival rates of cryopreserved porcine embryos collected on Day 6 (Day 0=onset of estrus) with various cryprotectants. Eighty two embryos at different stages, ranged from expanded blastocyst to hatched blastocyst, were allocated to 6 experimental groups in different combinations of cryoprotectants glycerol, egg yolk and/or trehalose. Porcine embryos were cryopreserved using conventinal slow freezing precedures. The embryos were equilibrated with one of the freezing solutions, cooled from 25 to -7$^{\circ}C$ at 1$^{\circ}C$/min, seeded at -7$^{\circ}C$ frozen to -36$^{\circ}C$ at 0.5$^{\circ}C$/min, and then plunged into liquid nitrogen. The frozen embryos were thawed by immersion in 37$^{\circ}C$ water and the cryoprotectants were removed by dilution with 0.5M sucrose solution. Embryonic survival was estimated from the normal development of embryos for 12, 24 and 48hrs culture. Then the embryos were stained and their cell nuclei was counted. The survival rates of morphological embryos were significantly higher in group I (10% glycerol) or group IV (10% glycerol＋10% egg yolk＋0.5M trehalose) than those in other groups, although the nuclei number was quite higher in embryos treated with 10% glycerol and 0.25M trehalose at expended blastocyst. However, hatched blastocysts showed higher viability and nuclei number treated with either egg york or trehalose, but the survival rates after 48hrs of culture were quite low. These results indicate that egg yolk and trehalos as a supplement to freezing solutions can be useful to the cryopreservation ofn porcine embryos.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• International Journal of Oral Biology
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• v.32 no.3
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• pp.109-112
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• 2007

Somatic cells nuclear transfer (SCNT) is a useful tool in studies of developmental biology and animal cloning. However, SCNT experiments only are allowed to skilled technical experts. In this experiment, laser-assisted zona pellucida piercing tool (LASER) was applied in murine SCNT. LASER minimized the use of piezo-driven micromanipulator (PIEZO), reducing chances of problems caused by PIEZO pulses. LASER reduced time that took to pierce zona pellucida in removal of nucleus from oocyte and somatic cell injection, which might have taken longer time with PIEZO. Time and difficulties that took researcher of equivalent skilled for their experiments were decreased with LASER, and this might affect the improvement of embryonic development. (LASER, 6.2% versus PIEZO, 2.9%; P<0.05). Thus, these data support that the use of LASER can be used for zona pellucida piercing in murine SCNT program as an alternative to PIEZO.

• Molecules and Cells
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• v.27 no.6
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• pp.635-640
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• 2009

Testis-derived germline stem (GS) cells can undergo reprogramming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.222-231
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• 2019

Among fatty acid families, the polyunsaturated fatty acids were demonstrated to be mediators in various reproductive processes as precursor of steroid hormone (via cholesterol) and prostaglandins (via arachidonic acid), and in the last decade, major research was focused on the effects of omega-6 and especially omega-3 fatty acid. Eicosapentaenoic acid, the longest members of omega-3 fatty acid family, can be produced by a series of desaturation and elongation reactions from shorter member such as α-Linolenic acid. However, very few studies have provided detailed descriptions of Eicosapentaenoic acid effects and mechanisms of action in mammalian oocytes. The purpose of this study was to evaluate the effect of Eicosapentaenoic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of Eicosapentaenoic acid was added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, nuclear maturation rate, blastocysts quality, and levels of prostaglandin E2, 17β-estradiol, progesterone in the spent medium. High doses (100 μM) of Eicosapentaenoic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E2 synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM Eicosapentaenoic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of E2/P4 also significantly increased compared with control group (p < 0.05). However, Supplementation of 100 μM Eicosapentaenoic acid showed high apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17β-estradiol/progesterone also significantly decreased compared with control group (p < 0.05). Our results indicated that supplementation with appropriate levels of Eicosapentaenoic acid beneficially affects the change of hormone synthesis for controlling oocyte maturation, leading to improved embryo quality. However, high doses of Eicosapentaenoic acid treatment results in detrimental effects.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.201-207
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• 2011

The present study was performed to investigate the survival and subsequent embryonic developmental rate of immature and mature oocytes after vitrification and pronuclear stage embryos after slow-freezing and vitrification. We have also tried to examine the dependency of concentrations (7.5, 15%) and exposure time (5, 10, 20 min) of ED cryoprotectant on developmental rate of pronuclear stage embryos. The developmental rates of 2-ce1l and blastocyst embryos at mature oocytes were significantly (p<0.05) higher than immature oocytes. After slow freezing, vitrification and thawing of pronuclear stage embryo, the survival and developmental rates of blastocysts and hatched blastocysts were significantly (p<0.05) higher after vitrification than after slow-freezing. On contrary, the developmental rates of 2-cell embryos were significantly (p<0.05) higher after slow freezing than after vitrification. The cryopreservation methods of pronuclear stage embryos vitrified by exposed to 7.5% ED solution for 5 minutes was significantly (p<0.05) higher than other experimental group. The results of our study suggest 1hat the developmental rates of mature oocytes have been more successful than immature oocytes during vitrification. Vitrification was more efficient than slow freezing in case of pronuclear stage embryos. The effective cryopreservation method of pronuclear stage embryos was vitrified by exposed to 7.5% ED solution for 5 minutes.