• 한국수정란이식학회지
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• 제18권2호
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• pp.151-156
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• 2003

한우의 수정란을 보존하기 위하여 고급육계통과 다유계통의 공란우를 선발하여 과배란처리에 따른 황체의 반응, 황체수에 대한 채란된 수정란의 수 그리고 채란 수정란수에 대한 이식가능한 수정란 생산에 미치는 영향을 조사하였다. 다유계통의 공란우는 이유시 체중에 대한 육종가 상위 20%이내, 고급육 계통의 공란우는 육질에 대한 육종가 상위 20%이내의 종빈우를 선발하였다. 과배란처리 방법은 발정후 11일차에 PEG 30% FSH를 견갑부에 피하주사로 1회 투여하고 13일차에 PGF$_2$a 25mg을 투여하였으며 15일차와 16일차에 각각 2회씩 인공수정하였고 23일차에 채란하였다. 과배란처리에 따른 황체의 반응은 좌측난소가 47.1% (242/513), 우측난소가 52.8%(271/513)로써 우측의 반응이 좋게 나타났고 황체의 반응과 비교하여 채란된 수정란의 회수율은 79.9%(410/513)로 나타났다. 회수된 수정란 중에서 이식이 가능한 수정란은 86.5%(355/410)이었고 수정란의 발달단계에 있어서 상실배는 61.6%(219/355), 배반포는 38.3%(136/355)로 나타났으며 두당 이식가능한 생산수는 6.4개였다.

• Clinical and Experimental Reproductive Medicine
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• 제28권1호
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• pp.41-46
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• 2001

Objective: To determine the effects of leukemia inhibitory factor (LIF) on embryonal development in in vitro culture. Methods: This is designed in vitro model using eggs from mouse. The eggs from mouse were assigned 29 for control group, 53 for 20 ng/ml of LIF, 88 for 40 ng/ml of LIF, 68 for 80 ng/ml of LIF respectively for in vitro fertilization. And 26 fertilized eggs at 2 cell stage from mouse also were assigned. The mouse embryos of all groups were cultured in medium supplemented with LIF in different concentrations, whereas the eggs in control group was cultured in medium without supplement of LIF. Results: At 72 hours culture of eggs from in vitro fertilization, there was a slight increas in rate of embryonal development to morula in both LIF-20 and LIF-40 as results of 64.15% and 75% respectively, while 42.65% in inferior rate of LIF-80, compare with 51.72% in control group. But the difference between these each groups were not significant in statistically ($p{\le}0.05$). And after 96 hours culture of eggs, the rates blastocyst formation was significantly higher in both LIF-20 and LIF-40 as 56.6% and 63.63% than those in control and LIF-80 as 44.83% and 35.29% respectively. On culturing eggs from in vivo fertilization, the rates of blastocyst formation was significantly not only higher as 85% and 81.81% respectively in medium supplemented with LIF-40 and LIF-80 than 42.3% in LIF-20 but also embryonal cell viability were remakedly improved at 96 hours after culture. Conclusion: The LIF in low dose is embryotrophic, but LIF in high dose is embryotoxic on eggs from in vitro fertilization. Whereas on culturing eggs from in vivo fertilization, LIF is more beneficial with dose dependent in high concentration.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.30-30
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• 2001

The culture of preantral follicles has important biotechnological implications through its potential to produce the large quantity of oocytes for embryo production, transgenesis research, conservation of rare breed, and a potential source of ovarian genetic material. The present study was conducted to establish the optimal conditions of in vitro culture for intact bovine preantral follicles; and to examine the developmental ability of oocytes derived from the in vitro-grown preantral follicles; and to investigate the effects of various concentrations of FSH and LH on these processes. Bovine preantral follicles (150 $\pm$ 1.2${\mu}{\textrm}{m}$), surrounded by theca cell, were isolated enzymetically and mechanically from ovarian cortical slides in Leibovitz L-15 medium containing 1 mg/$m\ell$ collagens and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium with insulin, transferrin, and selenite. The survival was tested by frypan Blue and Hematoxylin. The survival and growth rates of follicles were higher in FSH treatment groups than these in control (P<0.001), but there were no significant differences between the LH treatment groups and the control. In 25 days, the survival and growth rates of follicles in FSH and LH treatment group (50%, 300$\pm$1.0${\mu}{\textrm}{m}$) were higher than in FSH treatment group (40%, 244$\pm$0.5${\mu}{\textrm}{m}$) and the control group (25%, 160$\pm$ 1.0${\mu}{\textrm}{m}$). Fifty-five percent of healthy antral follicles were obtained, and 60% of the oocytes complete meiotic maturation to the metaphase II stage. Twenty-two percent of the mature oocytes underwent cleavage, and 9% developed to the blastocyst stage. In this study, in vitro-grown oocytes (111 $\pm$ $1.5mutextrm{m}$), under our culture conditions, were not equivalent in size to the in vivo-grown oocytes (130$\pm$1.3${\mu}{\textrm}{m}$). Therefore, these results suggest that bovine preantral follicles with intact theca cell can grow to the antral stage in 25days, and that oocytes from those follicles can acquire the meiotic competence and normally undergo fertilization and development to the blastocyst stage. However, the developmental capacity of in vitro-grown oocytes is presumably not comparable to those of the in vivo counterparts.

• Journal of Periodontal and Implant Science
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• 제27권2호
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• pp.329-346
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• 1997

Dental implants have been developed for enhancement of osseointegration. Biocompatibility, bone affinity and surface characteristics of dental implants are very important factors for osseointegration. The aim of the present study was to determine the cytotoxicity and the bone affinity of titanium phosphide(Ti-P) implant material. The Ti-P surface was obtained by vacuum sintering of titanium within compacted hydroxyapatite powder. The composition and the chemical change of the surface were determined by Auger electron spectroscopy. The in vitro cytotoxicity was evaluated by the viability of the bone cells and macrophages obtained from chicken embryo and rat,s peritonium, respectively. For the comparative evaluation, 316L stainless steel, commercially pure titanium and Ti-P materials, prepared in size of 1O.0mm in diameter and 5.0mm in height, were immersed separately in bone cells and macrophages for 10 days. For the evaluation of the in vivo bone affinity, 316L stainless steel, commercially pure titanium and Ti-P materials, prepared in size of 5.0mm in diameter and 10.0mm in length, were implanted after drilling in diameter 5.5mm in femurs of 2 dogs weighing 10Kg more or less. Six weeks after implantation the specimens were prepared for histopathological examination and were observed under light microscope. In comparison of in vitro bone cell viability, Ti-P and commercially pure titanium groups were not significantly different from control group (p>O.1), but 316L stainless steel group was significantly lower than control group(p<0.05). There was no statistical difference in the viability of macrophages between 3 different groups and control group(p>O.l). In comparison of in vivo study, 316L stainless steel and commercially pure titanium showed fibrous encapsulation, but Ti-P showed remarkable new bone formation without any fibrous tissue. The results demonstrate that Ti-P has favorable biocompatibility and bone affinity, and suggest that dental implants with Ti-P surface may enhance osseointegration.

• 한국수정란이식학회지
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• 제22권3호
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• pp.191-194
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• 2007

본 연구는 도축된 제주마의 난소를 이용하여 초음파 유도난포란 채취 기술을 확립하고자 난소, 난포 및 황체의 크기를 초음파상과 육안적 측정치를 비교하고자 하였다. 초음파상 측정치와 caliper를 이용한 실제 크기의 측정치 비교에서는 통계학적 차이는 보이지 않았다. 배란 직전의 난포를 조사한 결과, 초음파상에서 관찰한 것은 난소 한 개 당 평균 0.83개와 평균 크기는 2.86 m였으며, 육안으로 관찰된 것은 난소 한 개 당 평균 0.75개와 평균 크기는 2.3 cm였다. 난소 한 개 당 평균난포 수는 초음파상에서는 4.25개와 육안에서는 4.38개였다. 제주마 난소의 난포 크기 및 수를 초음파상과 육안으로 조사하였던 바, 유의적인 차이가 나타나지 않았다. 이러한 결과로 차후 초음파를 이용한 OPU와 말에서 번식 기술과 관련된 연구에 기초 정보가 제공되리라 사려된다.

• 한국수정란이식학회지
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• 제30권3호
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• pp.249-255
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• 2015

Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of ${\beta}-cells$, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from ${\alpha}$-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3~4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in ${\beta}-cell$ replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

• 한국발생생물학회지:발생과생식
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• 제3권1호
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• pp.39-44
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• 1999

성선자극호르몬으로 자극된 난소로부터 회수된 사람의 성숙난자의 전핵형성과 초기발생에 정자의 운동성과 성숙도가 미치는 영향을 조사하였다. 세포질내정자주입 (ICSI)은 HEPES-buffer mTCM-199 배양액에서 실시하였다. 체내에서 성숙된 난구세포부착난자를 ICS에 의하여 사정된 운동성 정자 또는 비운동성 정자로 수정하였을 때 운동성 정자로 수정된 난자가 비운동성 정자로 수정된 난자보다 전핵형성율이 높았다 (79.8% vs 51.7% ; p<0.002)). 그러나 체내에서 성숙된 난구세포부착난자를 ICS에 의해 정소 내 운동성 정자 또는 비운동성 정자로 수정하였을 때 운동성 정자와 비운동성 정자 사이의 전핵형성율은 차이가 없었다. 10.0 mM lactate, 0.5 mM pyruvate, 0.2 mM taurine, 1.0 mM glutamine, 2.22 mM MEM amino acids, vitamin 그리고 10% 사람 난포액이 포함된 수정 Tyrode 배지에서 전핵이 형성된 수정란의 초기 발생은 정자의 채취원과 운동성에 관계없이 9∼16세포기로 발생하였다.

• 한국가축번식학회지
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• 제19권2호
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• pp.81-88
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• 1995

Culture medium (ASF-301) of tHUE-2 cell, human endothelial cell line, and culture medium of these cells (conditioned medium : CM) which affect embryonic development of in vivo fertilized 1-cell embryos of mouse were examined. Two-cell stage block of mouse embryos was overicomed in ASF-301 and CM without EDTA, which usually added in basic medium (modified Whitten Medium: MWM, control) to overcome the 2-cell stage block. The developmental rates of embryos to the blastocyst stage were significantly increased in MWM containing 12.5% of growth factors added to ASF-301 (10mg/ $\ell$ transferrin, 1mg/$\ell$ insulin, 0.01mg/$\ell$ EGF) than those of 100% addition and control, 78.0% vs 20.8 and 52.3%, respectively (P<0.05), but the growth factors was not affected the hatching rate of blastocyst. Using ASF-301 or CM which was not treated, embryonic development into the blastocyst and hatched blastocyst stages were not affected. However, proportions of embryonic development into the blastocyst and hatched blastocyst stages were significantly higher in dilution (ASF-301 1:10; CM 1:3~1:6) than those in control (P,0.05). In ASF-301 dialyzed M.W.<10000 dialysis membrane, the developmental rate upto the hatched blastocyst stage was significantly increased, compared to ASF-301 which was not dialyzed (P<0.05), and hatching rate of blastocyst of these group was singnificantly increased than those in MWM (P<0.05). Compared to CM which was not dialyzed, however, in dialyzed CM was significantly decreased, compared to untreated CM (P<0.05), especially any hatched blastocyst was not appeared. As a result of these experiments indicated that a kind or porper treatment such as a dilution of complex synthetic cell culture medium and conditioned medium, and that a optimal concentration of growth factors are usuful for embryo cultrue in vitro.

• BMB Reports
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• 제39권5호
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• pp.469-478
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• 2006

Vascular endothelial growth factor (VEGF)-A, a major regulator for angiogenesis, binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). These receptors regulate physiological as well as pathological angiogenesis. VEGFR2 has strong tyrosine kinase activity, and transduces the major signals for angiogenesis. However, unlike other representative tyrosine kinase receptors which use the Ras pathway, VEGFR2 mostly uses the Phospholipase-$C{\gamma}$-Protein kinase-C pathway to activate MAP-kinase and DNA synthesis. VEGFR2 is a direct signal transducer for pathological angiogenesis including cancer and diabetic retinopathy, thus, VEGFR2 itself and the signaling appear to be critical targets for the suppression of these diseases. VEGFR1 plays dual role, a negative role in angiogenesis in the embryo most likely by trapping VEGF-A, and a positive role in adulthood in a tyrosine kinase-dependent manner. VEGFR1 is expressed not only in endothelial cells but also in macrophage-lineage cells, and promotes tumor growth, metastasis, and inflammation. Furthermore, a soluble form of VEGFR1 was found to be present at abnormally high levels in the serum of preeclampsia patients, and induces proteinurea and renal dysfunction. Therefore, VEGFR1 is also an important target in the treatment of human diseases. Recently, the VEGFR2-specific ligand VEGF-E (Orf-VEGF) was extensively characterized. Interestingly, the activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice with minor side effects such as inflammation compared with VEGF-A, suggesting VEGF-E to be a novel material for pro-angiogenic therapy.

• The Korean Journal of Physiology and Pharmacology
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• 제3권2호
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• pp.157-164
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• 1999

To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP $(400\;{\mu}M)$) or forskolin/isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5'-flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5'-flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5'-flanking region of the gene in vitro, and the regulatory mechanism may be important for in vivo regulation of AQP2 expression.