• 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.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.207-214
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• 1996

To examine the developmental capacity of manipulated embryos after ultrarapid refreezing and thawing, mouse embryos were biopsied at 4-cell stage, frozen twice at 4-cell and morula stages, respectively, and then transferred to rec-ipients. Single blastomeres were biopsied from 4-cell embryos by a modified aspiration method. Biopsied 4-cell embryos were equilibrated into freezing medium at room temperature for 2.5 min, loaded into 40 $\mu$I of freezing medium in 0.25 ml plastic straw and then directly immersed into liqiud nitrogen. Freezing medium for 4-cell embryos consisted of 4.0 M ethylene glycol and O.25 M sucrose in dPBS supplemented with 6 mg/lm BSA. Morulae were frozen into freezing medium containing 5.0 M glycerol instead of ethylene glycol. Thawing was conducted by agitating each straw in 3TC water for 20 sec. The c content of each straw was expelled into 0.5 ml of dilution medium, which consisted of 0.25 M sucrose and 3 mg/ml BSA in dPBS. The thawed embryos were rehydrated in dilution medium for 10 min, washed 3 times with dPBS and then cultured in M16 medium at 37$^{\circ}C$, 5% CO$_2$ in air. Blastocysts that developed from frozen or refrozne biopsied embryos were transferred to recipients on Day 3 of pseudopregnancy, respectively. In vitro and in vivo developmental rates of the biopsied and intact 4~cell embryos after freezing and thawing were 78 (10l/130) and 25% (10/40), and 91 (114/125) and 30% (12/40), respectively. Although the rates of in vitro development of biopsied and intact embryos to blastocyst stage were significantly different after freezing and thawing (P

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.11-17
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• 1998

This study was carried out to investigate the in vivo developmental potential of mouse zona-hatched blastocysts (HBs). The HBs were cultured in vitro until day 5 and day 6 from zygotes produced in vivo and classified to small (S-HBs), medium (M-HBs) and large (L-HBs) on the basis of embryo diameters. The results obtained in these experiments were summarized as follows ; 1) when the blastocysts at day 4 were further cultured for $24\sim48hr$, HBs obtained at day 5 and day 6 culture in vitro were 29.1% and 22.8%, respectively. 2) Also, when the total cell number of HBs were counted, cell numbers of classified HBs on day 5 and day 6 to small ($77.3\pm5.3$, $59.6\pm4.4$), medium ($83.7\pm4.0$, $66.8\pm3.5$) and large ($100.7\pm2.6$, $88.9\pm3.8$) were increased as their size increases. Especially, there were significantly different between S-HBs and L-HBs (p<0.01). 3) In addition, when the classified HBs were transferred into when the classified HBs were transferred into day 3 pseudopregnant recipients, the pregnancy and implantation rates of S-HBs (28.6%, 15.7%), M-HBs (44.4%, 30.9%) and L-HBs (62.5%, 49.1%) at day 5 were increased as their size increases. However, this pattern was not showed in embryo transfer of day 6 HBs. But, when the live fetuses formation against total implantation rates were observed, the result (87.5%) of S-HBs of day 5 was significantly higher than that of the others (p<0.01). Therefore, this study demonstrates that in vitro cultured healthy HBs can not only be developed normally with good pregnancy rates, implantation rates and live fetuses formation, but also served as a fundamental data for utility of supernumerary HBs in human blastocyst transfer.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.103-108
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• 1996

Through the previous studies(I,II), it was observed that human follicular fluid(HFF) was more effective than human fetal cord serum(HFCS) on promoting meiotic resumption of oocytes and improving embryonic development of mouse in vitro. On the basis of these results, we have gradually exchanged HFCS with HFF as protein supplement in human ART. This study was performed to investigate the efficiency of HFF on improving the pregnancy rate in ART. Oocytes were retrieved transvaginally from patients treated with pituitary suppression with GnRH-agonist and ovarian stimulation with human menopausal gonadotro-pin(HMG) and pure follicle stimulating hormone(FSH). Aspirated oocytes were rinsed and cultured in TCM-199 containing HFF, and the concentrations of HFF were adjusted to 10, 20, and 30% according to the use for insemination, embryo growth and embryo transfer, respectively. As possible as, we tried to do embryo transfer into fallopian tube to mimic the coincidence of the cell stage with the place of sojourn in vivo, so we performed various ART programs(IVF & ET; in vitro fertilization, ZIFT; zygote intra fallopian-tube transfer, ZIFT & ET) according to the tubal conditions of patients. On the while, intra cytoplasmic sperm injection(ICSI) was used to assist IVF of the patients who had shown poor standard IVF results by immunological or severe male factor. Of the 255 cycles of ART programs using HFF as protein supplement, 118 cycles were turn out to be succeeded in pregnancy(46.2%, per cycle, p<0.05), while 21 pregnancies were achieved in the 69 cycles using HFCS(30.4%). The 255 cycles using HFF were subdivided into cycles with the type of ART programs, and each pregnancy rate of the ART programs were 44.7% (IVF & ET, 76/170 cycles), 53.4%(ZIFT, 31/58 cycles) and 40.7% (ZIFT & ET, 11/27 cycles), respectively. In the 61 ICSI cycles using HFF, 28 cycles succeed in pregnancy(45.9%), while 7 pregnancies were obtained in the 17 ICSI cycles using HFCS. Also the ongoing pregnancy rate in the group using HFF(78.8%, 93/118 cycles) was higher than that in the group using HFCS(61.9%). Therefore, we found that the use of HFF as protein supplement was more suitable and effective than the use of HFCS to improve the pregnancy rate in ART.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.143-156
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• 2006

Major characteristics of embryonic stem cells (ESCs) are sustaining of sternness and pluripotency by self-renewal. In this report, transcriptional profiles of the molecules in the developmentally important signaling pathways including Wnt, BMP4, $TGF-\beta$, RTK, Hh, Notch, and JAK/STAT signaling pathways were investigated to understand the self-renewal of mouse ESCs (mESCs), J1 line, and compared with the NIH3T3 cell line and mouse embryonic fibroblast (MEF) cells as controls. In the Wnt signaling pathway, the expression of Wnt3 was seen widely in mESCs, suggesting that the ligand may be an important regulator for self-renewal in mESCs. In the Hh signaling pathway, the expression of Gli and N-myc were observed extensively in mESCs, whereas the expression levels of in a Shh was low, suggesting that intracellular molecules may be essential for the self-renewal of mESCs. IGF-I, IGF-II, IGF-IR and IGF-IIR of RTK signaling showed a lower expression in mESCs, these molecules related to embryo development may be restrained in mESCs. The expression levels of the Delta and HESS in Notch signaling were enriched in mESCs. The expression of the molecules related to BMP and JAK-STAT signaling pathways were similar or at a slightly lower level in mESCs compared to those in MEF and NIH3T3 cells. It is suggested that the observed differences in gene expression profiles among the signaling pathways may contribute to the self-renewal and differentiation of mESCs in a signaling-specific manner.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.76-76
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• 2002

본 실험은 Piezo-미세조작기(PrimeTech Ltd., Japan)를 사용하여 마우스 핵이식 후 재구축배를 CZB와 KSOM 두가지 배양액을 사용하여 체외배양성적을 비교 검토하였다. MII의 미수정란은 성숙한 4~5주령 B6D2Fl에 hCG 주사 후 14시간째에 과적 방법을 통해 난관의 팽대부로 부터 회수하였고, metaphase II chromosome-spindle complex와 최소량의 세포질을 내경이 10$\mu\textrm{m}$인 피펫으로 흡입하여 탈핵하였다. 핵이식에 사용된 난구세포(8-l0$\mu\textrm{m}$)는 3시간동안 12% PVP 에처리 하여 piezo-미세조작기를 이용하여 세포질에 세포의 핵을 직접 미세주입 하였다. 핵이식 후 생존한 재구축배는 2시간동안 배양한 후 10mM SrC1$_2$와 5$\mu\textrm{g}$/$m\ell$의 cytochalasin B가 첨가된 $Ca^{2＋}$-free CZB에서 6시간 활성화 처리하였다, 활성화 처리 후 위전핵이 관찰된 재구축란을 CZB 와 KSOM 배지에서 배양하면서 발달률을 비교하였고, 상실배 및 배반포배로 발달한 재구축배를 day 3 대리모에 이식하였다. 표 1에서 보는 바와 같이 재구축배의 2-cell로의 발달률에 있어서 KSOM이 CZB에 비하여 유의적으로 높게 나타났으며(P<0.05), 또한 4-cell과 상실배/배반 포배로의 발달률에 있어서도 KSOM이 CZB에 비하여 유의적으로 높은 발달률을 나타내었다(P<0.01). 또한 KSOM 배지에서 배양된 상실배/배반포배를 대리모에 이식한 경우에 11.5 d.p.c에 생존한 태아가 관찰되었다. 이상의 결과로 핵이식 재구축배의 활성화 처리 후의 발생에는 KSOM 배지가 CZB 배지에 비하여 유효함을 확인 할 수 있었다.그와 같은 배양 기술을 이용하여 외래유전자를 도입한 일련의 결과에 관하여 보고 하고자한다., 이것은 세포내 유전자가 transfection되지 않은 세포도 neo selection에서 선발된다는 것을 제시하고 있다. 따라서 체세포를 이용한 형질전환동물 생산을 위해서는 세포내 유전자 도입과 선발 과정에서 나타난 colony에 대하여 보다 엄격한 screen을 하는 것이 필요한 것으로 생각된다.로 우점하였다. 여름철 식물플랑크톤 대발생에 영향은 수온과 직산염이 중요하였으나, 부유물질 크게 기여하지 못하였다.애를 확인하고 지도 관점을 파악하는 것을 포함한다. 그러나 본 논문은 역사발생적 수학 학습-지도 원리의 실제적인 적용에 관하여는 기초적인 연구에 지나지 않기 때문에, 역사발생적 원리를 학교수학에 실제적으로 적용하기 위해서는 각각의 내용에 대한 철저한 역사적 분석을 바탕으로 하는 후속 연구가 필요하다./TEX>구성교육${\lrcorner}$이 조선총독부의 관리하에서 실행되었다는 것을, 당시의 사범학교를 중심으로 한 교육조직을 기술한 문헌에 의해 규명시켰다.nd of letter design which represents -natural objects and was popular at the time of Yukjo Dynasty, and there are some documents of that period left both in Japan and Korea. "Hyojedo" in Korea is supposed to have been influenced by the letter design. Asite- is also considered to have been "Japanese Letter Jobcheso." Therefore, the purpose of this study is to look into the origin of the letter designs in t

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.379-384
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• 1997

Superovulation with exogenous gonadotropins creates a spectrum of pre or periovulatory hormonal changes with subsequent detrimental effects on oocyte quality, fertilization, embryo development, implantation and maintenance of pregnancy. Our recent study determined potential roles for insulin-like growth factor-1 (IGF-1) in uterine environment regulation and preimplant tation in the rat. The evidence indicates that IGF-l may play an important role in the main tenance of a receptive uterine environment for embryonic development and the regulation of decidualization. Embryonic loss and failure of implantations following superovulation may be partially attributed to disturbances in uterine IGF-l action as observed in this study. We investigated the effects of superovulatory doses of gonadotropins on frequency of chromosomal a abnormalities of mouse embryos. Chromosome a analysis of mouse zygotes and 8- to 16-cell stage embryos from spontaneously ovulated, 5, 10, and l 15 lU pregnant mare serum gonadotropin (PMSG) superovulated mice was carried out. Aneuploidy, polyploidy and structural chrom- osomal abnormalities were detected among the four groups. However, only polyploidy was correlated with superovulation. In 10 and 15 IV PMSG treated groups, the rate of polypoidy was 2.9% and 10.5%, respectively. Furthermore, there was a dose reponse relationship between the PMSG dose and the incidence of embryonic p polyploidy (P

• Development and Reproduction
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• v.6 no.1
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• pp.55-66
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• 2002

Embryonic stem cells(ES cells) are derived from the inner cell mass(ICM) of blastocysts, which have the potentials to remain undifferentiated, to proliferate indefinitely in vitro, to differentiate into the derivates of three embryonic germ layers. ES cells are an attractive model system for studying the initial developmental decisions and their molecular mechanisms during embryogenesis. Additionally, ES cells of significant interest to those characterizing the various gene functions utilizing transgenic and gene targeting techniques. We investigated the effects of reproductive hormones, gonadotropins(GTH) and steroids on the induction of differentiation and expressions of their receptor genes using the newly established mouse ES cells. We collected the matured blastocysts of inbred mice C57BL/6J after superovulation and co-cultured with mitotically inactivated STO feeder cells. After 5 passages, we confirmed the expression alkaline phosphatase(Alk P) activity and SSEA-1, 3, 4 expressions. The protocol devised for inducing ES differentiation consisted of an aggregation steps, after 5 days as EBs in hormone treatments(FSH, LH, E$_2$, P$_4$, T) that allows complex signaling to occur between the cells and a dissociation step, induced differentiation through attachment culture during 7 days in hormone treatments. Hormone receptors were not increased in dose-dependent manner. All hormone receptors in ES cells treated reproductive hormones were expressed lower than those of undifferentiated ES cell except for LHR expression in E$_2$-treated ES cells group. After hormone induced differentiation, at least some of the cells are not terminally differentiated, as is evident from the expression of Oct-4, a marker of undifferentiated. To assess their differentiation by gene expression, we analyzed the expression of 7 tissue-specific markers from all three germ layers. Most of hormone-treated group increased in the expression of gata-4 and $\alpha$ -fetoprotein, suggesting reproductive hormone allowed or induced differentiation of endoderm.

• Development and Reproduction
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• v.13 no.2
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• pp.89-95
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• 2009

p63 has been demonstrated to localize in stem cells and precursor cells of various epithelial tissues previously, but the localization of p63 throughout tooth formation, particularly during the enamel and root formation stages, remains to be adequately characterized. Therefore, in this study, we have demonstrated, via immunohistochemical methods, that p63 is ubiquitously expressed in the dental epithelium during tooth development. p63 was detected in the basal and suprabasal layers of the epithelia, including the skin, hair follicles, oral mucosa, and submandibular ducts. However, in the tooth region, all cells of the dental lamina, enamel organ, Hertwig's epithelial root sheath (HERS), and epithelial cell rests of Malassez (ERM) evidenced immunoreactivity for p63. These results indicate that p63 may perform different roles, other than stem cell maintenance, in tooth development.

• Journal of Pharmacopuncture
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• v.11 no.1
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• pp.127-141
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• 2008

Objectives : The purpose of this study is to investigate the effects of Chowiseungcheng-tang extracts on the preadipocytes proliferation in 3T3-L1 cell line, lipolysis of adipocytes in rat's epididymal adipocytes and localized fat accumulation of porcine by extraction methods(alcohol and water). Methods : Diminish preadipocytes proliferation and promote lipolysis of adipocytes do primary role to reduce obesity. So, we used 3T3-L1 mouse embryo fibroblasts(preadipocytes) and rat epididymal adipocytes from Sprague-Dawley rats to investigate the effects of Chowiseungcheng-tang extracts on the preadipocytes proliferation, lipolysis of adipocytes. They were treated with 0.01, 0.1, $1.0mg/m{\ell}$ Chowiseungcheng-tang alcohol and water extracts. And for the purpose of investigating the effects of Chowiseungcheng-tang alcohol and water extracts on the localized fat accumulation, we injected 0.1, 1.0, $10.0mg/m{\ell}$ Chowiseungcheng-tang extracts to porcine fat tissues and observed histological changes of them. Results : Following results were obtained from the preadipocytes proliferation and lipolysis of adipocytes and histological investigation of fat tissues. 1. Chowiseungcheng-tang extracts suppressed preadipocytes proliferation on the high dosage(especially $1.0mg/m{\ell}$), and especially alcohol extracts had better effects. 2. The alcohol extracts of Chowiseungcheng-tang decreased the activity of glycerol-3-phosphate dehydrogenase (GPDH) on the concentrations of 0.1, $1.0mg/m{\ell}$. Alcohol extracts had better effects than water extracts. 3. Chowiseungcheng-tang extracts increased lipolysis of adipocytes on the concentrations of 0.1, $1.0mg/m{\ell}$, and especially on the concentration of $1.0mg/m{\ell}$ alcohol extract of Chowiseungcheng-tang had better effect. 4. The water extract of Chowiseungcheng-tang had significant activity to the destruction of porcine fat cell membranes only on the concentration of $10.0mg/m{\ell}$, but alcohol extracts of Chowiseungcheng-tang had it on all concentrations. Conclusions : The alcohol extracts of Chowiseungcheng-tang had much better effects on the preadipoeytes proliferaton, lipolysis of adipocytes and localized fat accumulation than water extracts.