• 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.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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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.

• Korean Journal of Plant Resources
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• v.8 no.3
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• pp.237-246
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• 1995

In this study, the callus was induced and regenerated from the immature embryo and ultrastructural characteristics of developmental stages in Citrus junos SIEB, were investigated. The yellowish callus was induced by 5 to 6 week of culture of citrus. In proliferation callus after 6 weeks of culture, large vacuole was formed by fusion between adjacent small ones. In the non-embryogenic callus cultured for 12weeks, re-differentiated cells of callus showed the large nucleus with globular nucleus and amyloplast with large size of starches. In the embryogenic callus cltured for 14-16 weeks, the active exocytosis occurred in cells, secretory vesicles appeared on cell membrane and small particles from cytoplasm were released to intercelluar space. In the embryogenic callus cultured for 24 weeks, a sperical type of chloroplast bounded on cytoplasm by double membrane and typical grana was dispersed equally among matrix. In the normal plantlet after 26 weeks of culture, a lot of vessels and companion cells apperaed in the leaf cell of plantlet. In the normal plantlet after 30 weeks of culture, the immature leaf showed many small companion cells, sieve tubes and central vacuole. Also, the secondary vacuole protruded into the central vacuole and elongated chloroplasts near plasma membrane. In the matured plant habituated on the soil, palisada tissue composed of orderly arranged cells contained the nucleus in the center of the cell and large vacuoles on either side of the nucleus.

• 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.24 no.1
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• pp.101-109
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• 1997

This study was carried to determine the possibility of finding motile spermatozoa and fertilization, pregnancy rate after testicular sperm extraction(TESE) with ICSI in obstructive and non-obstructive azoospermic patients. In 154 cases(132 patients), obstructive azoospermia was 77 cases and non-obstructive azoospermia was 77 cases. In obstructive azoospermia, patients generally showed normal spermatogenesis and included vas agenesis(n=8), multiple vas obstruction(n=7), epididymal obstruction (n=54). Total of 982 retrieved oocytes were obtained and 84.4% were injected. The fertilization rates with 2 PN and cleavage rate were 72.5% and 62.3%, respectively. 30 pregnancies(38.9%) were achieved and the ongoing pregnancies were 22 cases (28.6%). In non-obstructive azoospermia, patients showed hypospermatogenesis(n=49), maturation arrest(n=4), Sertoli cell only syndrome (n=24). The various stages of spermatogenic cell could be retrieved by TESE and could be reached normal fertilization and embryo development with ICSI. Total of 1072 retrieved oocytes obtained and 80.2% were injected. The fertilization rates with 2 PN and cleavage rate were 52.8% and 68.9%, respectively. 22 pregnancies(30.1%) were achieved and the ongoing pregnancies were 19 cases(26.0%). Conclusively, the combination of TESE with ICSI using testicular spermatozoa can achieve normal fertilization and pregnancy rate and effective method in obstructive and non-obstructive azoospermic patients.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.1
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• pp.36-40
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• 2007

Proteins and lipids not only provide a source of energy to the cell, but also play vital roles in modifying the physical properties and function of the biological membranes. In the present study, we investigated the biochemical constituents, viz. proteins and lipids, in growing oocytes of goat antral follicles during summer and winter seasons. Goat genitalia in phosphate buffered saline (pH 7.4) were brought to the laboratory within one hour of slaughter under aseptic conditions at $37^{\circ}C$. Oocytes were aspirated from normal small (<3 mm in diameter) and large (>3 mm) follicles and pooled for biochemical estimations. A significant increase in the amount of protein and lipid was observed with the growth of the oocyte. The amount of protein varied non-significantly with the season, while the amount of lipid varied significantly. The amounts of phospholipid, cholesterol, free fatty acid, and triglyceride increased with the growth of the oocyte, but no significant effect of season in these constituents was observed. Lysolecithin, sphingomyelin, and sterols were the polar lipids identified in both oocytes prepared from small follicles (small oocytes) as well as large follicles (large oocytes). In addition, the small oocytes also contained phosphatidyl serine, while large oocytes contained phosphatidyl glycerol phosphate and phosphatidyl inositol. Among non-polar lipids, triglycerides and long chain alcohols appear only in small oocytes and not in large oocytes. Monoglycerides, 1,2-diglycerides, 1,3-diglycerides and o-dialkyl glycerol ethers, fatty acids, fatty acid methyl esters, and wax esters were identified in both small and large oocytes. Information on biochemical composition of growing oocytes is relevant to oocyte and embryo competence, culture and cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.109-114
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• 1996

General PCR technique alone has a limitation for preimplantation genetic diagnosis(PGD) using single blastomere. Recntly developed primer extension preamplification(PEP) technology amplifies the whole genome and thus, simultaneous multiple locus analysis became possible. In this study, we report the efficacy of PEP-PCR for PGD in three muscular dystrophy carriers undergoing IVF-ET. A total of 37 blastomeres were obtained from 40 embryos at six to eight cell stage in three IVF cycles in two DMD and one BMD carriers. Whole genome from single blastomeres were amplified using I5-base oligonucleotide random primers. PCR amplified products of exon 45 in the dystrophin gene and alphoid X/Y loci for gender determination were analysed by 2% metaphor gel electrophoresis. A total of 37 PEP-PCR replicates from 37 single blastomeres from 40 embryos and 37 blanks were performed. We obtained the reliable results for exon 45 and alphoid X/Y. Transfer of female embryos and unaffected male embryo was attempted in three couples. Unfortunately, pregnancy was not achieved in these cases. PEP-PCR is a reliable and efficient PGD method in multiple locus analysis using single blastomere.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.1-7
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• 1995

This study was accomplished to illuminate factors of contamination of microbes in the culture medium and effect of antibiotics on prevention of contamination in the medium when bovine follicular oocyte was matured, fertilized and developed in vitro. 1. When washed or unwashed semen diluted with TCM 199 was incubated for 24∼72hr, contamination was come out. 2. When diluted semen with TCM 199 which has penicillin, streptomycin, gentamycin, kanamycin or nystatin was incubated for 24∼72hr, contamination was not come out only in kanamycin. 3. When imported semen which was diluted in TCM 199 with penicillin, streptomycin, gentamycin, kanamycin or nystatin was incubated for 24∼72hr, contamination was not come out in all treatments. 4. When semen which was diluted in BO, CZB, Ham's F10 or TCM 199 was incubated for 24∼72hr, kanamycin showed no contamination in all treatments, but gentamycin showed contamination in CZB, Ham's F10 and TCM 199. 5. When the semen diluted in BO was moved at 24hr after incubation into BO and incubated for 72hr, contamination was not come out, but when it was moved into the TCM 199 and incubated for 72hr, contamination was come out at 48 to 72hr of incubation. 6. When the semen diluted in BO, BO＋BSA or BO＋FBS containing gentamycin, kanamycin or nystatin was incubated for 24∼72hr, the diluted semen in BO or BO＋BSA showed no contamination in all antibiotics but the diluted semen in BO＋FBS showed no contamination only in kanamycin. 7.The Pseudomonas cepacia, Serratia liquefaciens, Klebsiella pneumaniae was respectively isolated in the semen of A, B, and C bull and the microbes are highly affected by amikacin, tobramycin and kanamycin. 8. When bovine folicular oocyte was in vitro matured, fertilized and developed in the simple medium with kanamycin, 26.6% was developed to over 32cell stage embryo.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.111-111
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• 2003

Telomeres are the end of chromosomes and consist of a tandem repeat sequence of (TTAGGG)n and associated proteins. Telomerase is a ribonucleoprotein which act as a template for the synthesis of telomeric DNA. Telomeres are essential for chromosome stability and are related with cell senescence, apoptosis and cancer. Even though telomeres and telomerase have been studied extensively, very little is known about telomere dynamics in embryonic cells. This study was carried out to analyze the telomeres distribution and telomerase activity of chicken cells during embryonic and developmental stages. The target cells for analysing were sperms, ovulated ova, early embryonic cells and the cells from brain, heart, liver, kidney and germinal tissue in fetus. Telomeres distribution on target cells was analyzed by Q-FISH (Quantitation-Fluorescence in situ Hybridization) techniques using a chicken telomere repeat probe. Telomerase activity was performed by TRAP assay (Telomeric repeat Amplification Protocol) with target DNA. In results, the telomeres of chicken were found at the ends of all chromosomes. In addition, chicken had interstitial telomeres on chromosomes 1, 2 and 3. Telomerase activity was highly detectable in early embryonic cells, germinal tissues and kidney cells. Whereas telomerase activity was gradually down-regulated when the organs, including brain, heart, and liver, were developed from embryos. In the distribution of telomeric DNA on the embryonic and developmental stages, most of the cells was gradually decreased in telomere quantity during ontogenesis.