• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.315-323
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• 1994

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.77-81
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• 1994

To improve in vitro embryonic cell development, this study was desigend to culture in vitro fertilized early embryos of mouse in two different systems; conditioned medium alone and ampullary cells co-culture. Thirty two of 83 embryos(38.6%) were blocked in the 2 cell stage by co-culture, as compared to forty of 42 embryos(95.2%) in control group for 24hours culture. And all the embryonic cells cultured for conditioned medium alone were blocked for 48 hours culture. Twenty seven of 46 embryos (58.7 %) which overcome culture block in 2 cell stage by cocultured were developed morular and expanded blastocyst, and ninteen of 46 embryos(26.1 %) underwent hatching for 96 hours culture. The cellular fragmented rates for embryo were 26.2% in medium alone; 10 fragmented blastomere were graded mild status and 1 fragmented blastomere in severe status. On the other hand, the fragmented rate for 48 hours co-cultured were 15.7%03/83); 8 fragmented embryos were graded mild status, moderate status in 3 fragmented embryos and severe in 2 fragmented embryos respectively. In conclusion, the co-culture of embryos with ampullary cells is good to improve quality of embryos and overcome of culture block as well as development of cell cleavage.

• Development and Reproduction
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• v.2 no.2
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• pp.197-203
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• 1998

DNA methylation seems to play an important regulatory role in gene expression and cell differentiation during postimplantation embryonic development. However, the significance of DNA methylation which is maintained by the DNA MTase during preimplantation embryonic development, is not fully understood. In order to study the role of DNA methylation in the preimplantation embryos, the expression of DNA MTase transcripts was monitored in the oocytes and preimplantation embryos. The mRNA of DNA MTase was detected in the oocytes and pleimplantation embryos. The relative mRNA levels of DNA MTase were high from the stages of GV-oocytes and pronuclear embryos, and thereafter decreased gradually. By the treatment of $\alpha$-amanitin, it was confirmed that the transcripts presented in pronuclear embryos was derived from the maternal genome. The presence of transcripts of DNA MTase in the oocytes and pronuclear embryos suggests that the maintenance of DNA methylation may be necessary and seems to play an important role in gene expression and cell differentiation during preimplantation embryonic develop-ment in mouse.

• Development and Reproduction
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• v.5 no.2
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• pp.101-106
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• 2001

Present study was aimed to verify the role of insulin and TNF-$\alpha$ in development of preimplantation embryos. Mouse morula were cultured for 40 hr in the presence or absence of insulin(400 ng/ml) and TNF-$\alpha$ (50 ng/ml). The morphological development, cell number of blastomeres per blastocyst, and mitogen activated protein kinase(MAPK) activity were examined. The developmental rate and cell number per embryo were the highest in insulin treatment group and the lowest in TNF-$\alpha$ treatment group. There was no significant difference in developmental rate between control and insulin plus TNF-$\alpha$ group. Taken together, it suggested that TNF-$\alpha$ impaired embryonic development and that insulin rescued developmental impairment imposed by TNF-$\alpha$. In blastocysts, insulin treatment significantly increased MAPK activity. TNF-$\alpha$ decreased the MAPK activity in a concentration-dependent manner. In the TNF-$\alpha$(50 ng/ml) -primed embryos, activation of MAPK by insulin was attenuated. In conclusion, these results suggest that there was a cross talk between insulin and TNF-$\alpha$ by means of activation of MAPK in preimplantation embryos and that insulin might rescue damage of embryos exposed to TNF-$\alpha$.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.69-75
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• 2003

The objective of this study is to improve the efficiency of embryo transfer in generation of transgenic mice using inbred mouse (C57BL/6J) embryos. The embryos of C57BL/6J and BCF1 mice were superovulated by the standard protocol. One-cell stage of embryos were microinjected and the resulted one-or two-cell were transferred into one-or two-side oviducts of foster mother, respectively. When one-cell stage of embryos were transferred into one-side oviduct of 0.75 d.p.c. foster mother, the results were not ideal because of showing pregnancy ratios of 68.8$\pm$7.83% for C57BL/6J and 48.3$\pm$14.22% for BCF1, and development ratios of pups vs transferred embryos of 11.9$\pm$5.51% for C57BL/6J and 10.5$\pm$8.03% fur BCF1 . However, when two-cell stage of embryos were transferred into two-side oviducts of 0.5 d.p.c. foster mother, we got better results of 94.4$\pm$9.64% and 100$\pm$0% pregnancy ratio, and 22.1$\pm$0.4% and 21.8$\pm$0.38% development ratio for C57BL/6J and BCF1, respectively. Therefore, transferring two-cell stage of C57BL/6J embryos into two-side oviducts of 0.5 d.p.c. foster mother may be competitive to the result in BCF1 embryos. Even if there are a lot of other factors affecting these results, we conclude from these experiments that transfer of two-cell embryos into two-side oviducts of 0.5 d.p.c. foster mother is a more efficient and safe method than others in generating transgenic mice using inbred mice embryos.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.221-228
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• 2014

Despite that porcine spermatogonial stem cells (pSSCs) have been regarded as a practical tool for preserving eternally genetic backgrounds derived from pigs with high performance in the economic traits or phenotypes of specific human diseases, there were no reports about precise definition of niche conditions promoting proliferation and maintenance of pSSCs. Accordingly, we tried to determine niche conditions supporting proliferation and maintenance of undifferentiated pSSCs for short-term. For these, undifferentiated pSSCs were progressively cultured in different composition of culture medium, seeding density of pSSCs, type of feeder cells and concentration of growth factors, and then total number of and alkaline phosphatase (AP) activity of pSSCs were investigated at post-6 day culture. As the results, the culture of $4{\times}10^5$ pSSCs on mitotically in activated $2{\times}10^5$ STO cells in the mouse embryonic stem cell culture medium (mESCCM) supplemented with 30 ng/ml glial cell line-derived neurotrophic factor (GDNF) was identified as the best niche condition supporting effectively the short-term maintenance of undifferentiated pSSCs. Moreover, the optimized short-term culture system will be a basis for developing long-term culture system of pSSCs in the following researches.

• Journal of Embryo Transfer
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• v.8 no.2
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• pp.75-82
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• 1993

To investigate the effect of EDTA on the in vitro development of blastomeres isolated from 2, 4, and 8-cell embryos(termed 1 /2, 1 /4 and 1 /8 blastomeres, respectively) of ICR strain mice, those were cultured in vitro in 35 mm culture dishes containing NaHCO$_3$-BMOC-3 medium supplemented with 10, 50, 100, or 500 $\mu$M of EDTA at 37$^{\circ}C$ for 72hrs. under the atmosphere of 5% $CO_2$and 95% air. EDTA supplementation of 10, 50, or 100 $\mu$M to medium significantly(P<0.01) increased blastocyst formation rate compared with controls in 1 /2(58.3, 63.7, and 61.3% vs 21.6%), 1 /4(54.7, 57.5 and 62.2% vs. 2L3%), and 1 /8 blastomeres(46.2, 48.7, and 57.7% vs. 19.1%). Whereas, it was significantly(P<0.01) decreased to 4.5, 2.3, and 2.0% for 1 /2, 1 /4 and 1 /8 blastomeres, respectively by the EDTA supplementation of 500 M Both the nuclear number(P<0.05) and diameter of blastocysts(P<0.01) developed from balstomeres were significantly affected by the origin of blastomeres. The nuclear number of blastocysrs developed from 1/2, 1/4, and 1/8 blastomeres ranged 28.3i1.3, 24.18i1.2, and 19.84$\pm$0.9, respectively. And the diameter of those blastocysts was 87.2$\pm$1.1, 56.4$\pm$0.9, and 39.2$\geq$0.8 $\mu$M, respectively.

• 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.

• Biomedical Science Letters
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• v.15 no.4
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• pp.319-326
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• 2009

Extracellular ATP elicits diverse physiological effects by binding to the G-protein-coupled P2Y receptors on the plasma membrane. In addition to the short-term effects of extracellular nucleotides on cell functions, there is evidence that such purinergic signalling can have long-term effects on cell proliferation, differentiation and death. The 3T3-L1 cell line derived from mouse embryo is a well-established and commonly utilized in vitro model for adipocytes differentiation and function. However, the distributions and roles of P2Y subtypes are still unknown in the preadipocyte. In this study, we identified the distributions and roles of P2Y subtypes in preadipocyte using $Ca^{2+}$ imaging and realtime PCR. ATP increased the $[Ca^{2+}]_i$ in a concentration-dependent manner. ATP increased $Ca^{2+}$ in absence and/or presence of extracellular $Ca^{2+}$. Suramin, non-selective P2Y blocker, largely blocked the ATP-induced $Ca^{2+}$ response. U73122, a PLC inhibitor, completely inhibited $Ca^{2+}$ mobilization in 3T3-L1 cells. The mRNA expression by realtime PCR of P2Y subtypes was $P2Y_2:P2Y_5:P2Y_6=1.0:12.5:0.3$. In conclusion, we showed that $P2Y_5$ receptor is a dominant purinergic receptor in preadipocytes, and multiple P2Y receptors could involve in differentiation and migration via regulating of intracellular calcium concentration.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.131-138
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• 2001

This experiment was designed to evaluate effects of nonspecific immunostimulator(NIS) Barodon-FX(equation omitted), anionic alkali mineral complex and far-infrared radiation solution on in vivo-produced mouse and in vitro-produced bovine embryos to blastocyst development. Proportion of mouse embryos developing into blastocyst was not greater in BSA- and Barodon-added medium than in BSA-control, but there was signifcantly different(P ＜ 0.05) in hatching and hatched blastocyst development between 0.25% Barodon-and PVP-contained medium(54.7%) than PVP-control(32.5%). BOEC and GC resulted in higher proliferation rate(24∼40% and 17∼22%, respectively) in 0.25∼0.5% Barodon-added medium than in controls, but proliferation of GC and CC greatly decreased in 1∼2% Barodon-added medium. Effect of Barodon on cell proliferation greatly varied among somatic cells. Proportion of early bovine embryos developing into morula and blastocyst was significantly greater(P ＜ 0.05) in 0.5% Barodon-added medium(50% and 63.6%) than in control(31.6% and 27.4%) under co-culture with BOEC and GC, but developmental rate was not different between other Barodon treatments and control. These data indicate that effect of Barodon on cell proliferation significantly varied between somatic cells and that addition of 0.5% Barodon in BOEC-coculture system may further improve blastocyst development in early bovine embryos.