• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.75-75
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• 2002

The present study was conducted for the production of transgenic cloned cows by somatic cell nuclear transfer (SCNT) that secrete human prourokinase into milk. To establish an efficient production system for bovine transgenic SCNT embryos, the offset was examined of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos. An expression plasmid far human prourokinase (pbeta-ProU) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected using a lipid-meidated method. In Experiment 1, developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In Experiment 2, the effect of cellular senescence in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed. In Experient 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 ${\mu}{\textrm}{m}$) or small cell (<30 ${\mu}{\textrm}{m}$)] were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.

• Korean Journal of Animal Reproduction
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• v.14 no.4
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• pp.253-261
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• 1990

This study was carried out to investigate the factors affecting development in vitro of follicular oocytes fertilized in vitro in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follciles of 3~6mm. The bovine oocytes were matured in vitro for 20~24 hours in TCM0-199 containing 10% FCS and hormones (0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml estradiol-17$\beta$). The matured oocytes were fertilized in vitro using Percoll-separated frozen-thawed spermatozoa in BO solution containing caffeine(5mM) and heparin(10$\mu\textrm{g}$/ml). Twenty-four hours after insemination, the oocytes were cultured in vitro and then the effects of cumulus cell layer, co-culture with cumulus cells, bovine oviduct epithelial cells from ampulla or isthmus on development of ova, were studied. The results obtained are summarized as follows : 1. The in vitro development degree of oocytes attached with compact and dense layered cumulus cells was higher than that with 3~4 layered cumulus cells to be 9~16cells(P<0.01). 2. When the in vitro fertilized oocytes were co-cultured with bovine oviduct epithelial cells or cumulus cells, the development rate to be morula was 20.2% and 12.7%, respectively and the rates were higher than that of control, 2.1%(P<0.05). 3. The development rate to be morula was 15.8% and 23.8%, respectively when the in vitro fertilized oocytes were co-cultured with bovine oviduct epithelial cells from ampulla or isthmus, and the rates were higher than that of control, 0%(P<0.05%).

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.95-99
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• 2004

This study was carried out to assess whether the addition of myo-inositol to maturation medium could improve porcine oocyte maturation in vitro. Oocytes were cultured for the first 22 h in Witten's medium containing 10IU/$m\ell$ PMSG, 10 IU/$m\ell$ HCG supplemented with or without myo-inositol. Subsequently, they were cultured for additional 22 h in Witten's medium without hormone supplemented with or without myo-inositol. When the porcine oocytes were cultured in maturation medium containing myo-inositol, the proportion of metaphase II oocytes 44h after culture was higher in the myo-inositol group(P<0.05). To study effects of cumulus cell on the maturation induced by myo-inositol, we examined the maturation status of cumulus-enclosed or cumulus-denuded porcine follicular oocytes. The rates of maturation were significantly higher in the cumulus-enclosed oocytes(P<0.05). However, the maturation rates of cumulus-denuded oocytes cultured in medium containing myo-inositol were higher than those of control group(P<0.05). Our results suggest that myo-inositol may affect meiotic progression of porcine follicular oocytes and supplementation of myo-inositol in maturation medium may be useful for the in vitro maturation of porcine follicular oocytes.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.287-296
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• 2018

Ganglioside GM3 is known as an inhibition factor of cell differentiation and proliferation via inhibition of epidermal growth factor receptor (EGFR) phosphorylation. Our previous study showed that the exogenous ganglioside GM3 reduced the meiotic maturation of porcine oocytes and induced apoptosis at 44 h of in vitro maturation (IVM). However, the role of ganglioside GM3 in the relationship between EGFR signaling and apoptosis during porcine oocyte maturation has not yet been studied. First, porcine cumulus-oocyte complexes (COCs) were cultured in the NCSU-23 medium with exogenous ganglioside GM3 according to maturation periods (non-treated, only IVM I: 0 - 22 h, only IVM II: 22 - 44 h and IVM I & II: 0 - 44 h). We confirmed that the proportion of germinal vesicle breakdown (GVBD) increased significantly in the IVM I treated group than in the control group. We also confirmed that the meiotic maturation until M II stage and polar body formation decreased significantly in the only IVM I treated group. Cumulus cell expansion and mRNA levels of the expansion-related factors (HAS2, TNFAIP6 and PTX3) decreased significantly in the IVM I treated group than in the control group. Protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 decreased significantly in the GM3-treated groups, during the IVM I period. In addition, cellular apoptosis, determined using TUNEL assay, and protein levels of Cleaved caspase 3, were increased significantly in the GM3-treated COCs during the IVM I period. Based on these results, ganglioside GM3 exposure of porcine COCs during the IVM I period reduced meiotic maturation and cumulus cell expansion via inhibition of EGFR activity in pigs.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.1-7
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• 2007

This study was conducted to examine the role of intact cumulus cells during in vitro fertilization (IVF) on sperm penetration, male pronuclear (MPN) formation and subsequent embryo development of oocytes matured and fertilized in vitro. Cumulus-oocyte complexes obtained from the slaughtered gilt ovaries were matured for 44 h in TCM199 containing 10% porcine follicular fluid, epidermal growth factor and hormones. After maturation culture, denuded oocytes or oocytes with intact cumulus cells were coincubated with frozen-thawed boar semen for 8h in a modified tris-buffered medium containing 5mM caffeine and 10mM calcium chloride. Putative zygotes were fixed and examined for sperm penetration and MPN formation (Experiments $1{\sim}3$), or cultured in North Carolina State University-23 medium fo. 156 h (Experiment 3). In Experiment 1, sperm penetration was examined after insemination of denuded oocytes and oocytes with intact cumulus cells at the concentration of $7.5{\times}10^5$ sperm/ml. Optimal sperm concentration for IVF of cumulus-intact oocytes was determined in Experiment 2 by inseminating intact oocytes with $2{\sim}5{\times}10^6$ sperm/ml. In Experiment 3, denuded or intact oocytes were inseminated at the concentrations of $7.5{\times}10^5$ and $4.0{\times}10^6$ sperm/ml, respectively, and in vitro embryo development was compared. Sperm penetration was significantly (p<0.01) decreased in cumulus-intact oocytes compared to denuded oocytes (35.2% vs. 77.4%). Based on the rates of sperm penetration and normal fertilization, the concentration of $4.0{\times}10^6$ sperm/ml was optimal for the IVF of intact oocytes compared to other sperm concentrations. The presence of intact cumulus cells during IVF significantly (p<0.05) improved embryo cleavage (48.8% vs. 58.9%), blastocyst (BL) formation (11.0% vs. 22.8%) and embryo cell number $(22{\pm}2\;vs.\;29{\pm}2\;cells)$ compared to denuded oocytes. In conclusion, these results suggest that intact cumulus cells during IVF inhibit sperm penetration but improve embryo cleavage, BL formation and embryo cell number of porcine embryos produced in vitro.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.1-6
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• 1992

These experiments were undertaken as a basic study to understand the role of cumulus cell on in vitro maturation and fertilization process with identifying the cumulus cell-secreted proteins. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and fast protein liquid chromatography(FPLC), the proteins of cumulus cells were identified. The results obtained in these experiments were summarized as follows ; 1. When the proteins secreted from cultured cell for 30 hours were separated by SDS-PAGE, there were a major band (>94,000) and other minor 2 bands with molecular weight ranging 30,000∼67,000 dalton. 2. In addition, the fractionations of these proteins by FPLC were idirectly shown that three bands were hyaluronic acid, chondroitin sulfate, and heparin.

• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.249-255
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• 1993

Canine follicular oocytes were used to establish a reliable system for maturation and fertilization in vitro. Ovaries were obtained from either slaughter house or hormone-primed bitches of mixed breeds. The oocytes were recovered by mincing the ovaries in M2＋BSA. Good quality of oocyte-cumulus complexes (OCCs) were selected and cultured in TCM 199 containing 15% fetal calf serum(FCS) for 24~56 h in an atmosphere of 5% CO2 at 39$^{\circ}C$. Maturation rate of follicular oocytes was >87% showing metaphase I. Unlike other domestic animals the cumulus expansion did not occur fully in canine OCCs although minimum expansion was found between the cumulus cells and corona radiata cells, the clear nuclear morphology was presented for the first time by rapid staining. The IVM system used in this study may be useful to obtain fully maturated metaphase I oocyte in dog.

• Journal of Korean Society for Atmospheric Environment
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• v.6 no.2
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• pp.176-182
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• 1990

A one dimensional cumulus cloud model has been developed to investigate the size distribution of hydrometeors at different heights and at different stages of cloud development. The model results show that the exponential distribution of droplet radius that was employed by many investigators can misinterpret the cloud dynamic fields during the life cycle of cumulus cloud. The results also show the bimodal distribution of cloud water mixing ratios at 25 $\mu$m and 645 $\mu$m in radius at 45 minutes of simulation time. The developed model can be applied in future on the parameterization of cloud microphysical processes and air pollution models.

• Journal of Genetic Medicine
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• v.18 no.1
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• pp.1-7
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• 2021

The oocyte quality is of great importance in infertility as it reflects the follicle developmental potential and further affects the embryo development, clinical pregnancy outcomes. The analysis of gene expression in somatic cells is an important study to better clinical in vitro fertilization (IVF) outcomes in embryo selection reflecting the appropriate communication between the oocyte and somatic cells. Specifically, somatic cell transcriptomic technology can help assess biomarkers of oocyte and embryo ability. The present article aims to overview the basic aspect of folliculogenesis and review studies involving changes in candidate gene expression of cumulus or granulosa cell related to clinical outcomes in human IVF.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.