• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.261-265
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• 1997

The objective of this study was to examine the effect of EGF to the in vivo development of mouse IVF/IVC embryo. The 2-cell embryos were cultured in medium (5-6 embryos/25 ${\mu}l$/drop) w/wo EGF (10 ng/ml) and day 4 blastocysts recovered from each treatment group were transferred into the uteri of recipients of pseudopregnant day 3. The results obtained in this experiment were summarized as follows: 1. When the effect of EGF to the in vitro development and cell number of blastocysts produced from the culture of 2-cell embryos in w/wo EGF was determined, those results of EGF treatment group showed not significant difference compared with control. 2. However, when the effect of EGF to the in vivo development of blastocysts recovered from each treatment group was examined, production of the normal fetus against transferred embryos in EGF treatment group (51.2%) was very higher than that in control group (31.1%), although total implantation was not significantly different between treatment group (control: 64.4%, EGF: 69.8%). Therefore, this result suggested that EGF can affect to the in vivo development of IVF/IVC embryos through the improvement of embryo quality, although EGF treated embryos showed not significant development rate compared with control.

• Journal of the Optical Society of Korea
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• v.13 no.3
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• pp.335-340
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• 2009

Ultrashort pulse laser drilling of polycarbonate track-etched membrane (pTEM) material was used to fabricate a mouse embryo cell trapping device. Holes with a diameter of $2{\mu}m$ to $5{\mu}m$ were fabricated on a $10{\mu}m$ thick membrane using a femtosecond laser with a 150 fs pulse width and 775 nm wavelength and multiple-pulse irradiation. In cell trapping tests, the overall cell occupancy of the machined holes in the fabricated pTEM was found to be more than 80%. The results of a single pulse and multiple pulse irradiation were compared in terms of the surface quality. It was generally found that a single pulse with high energy was less desirable than irradiation with multiple pulses of lower energy.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.287-292
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• 1998

The objective of this study was to test whether ZP drilling using a 1.48$\mu$m diode laser beam on mouse IVF embryos becomes effective the hatching and normal in vivo development, as a preliminary test for obtaining the additional proof that the 1.48$\mu$m diode laser could be used safely for human applications. The results obtained in this experiment were as follows: when the hatched rates of mouse embryos by laser ZP drilling according to the embryonic stage were examined until 72 hr (in case of blast tocyst: day 4 after IVF) or 120 hr (in case of 4-cell: day 2 after IVF) after treatment, the d data of laser drilled blastocysts (81.8%) was significantly higher than those of control (hatching blastocyst: day 4 after IVF) (54.2%) and laser drilled 4-cell embryos (45.5%) (p<0.05). When the effect of laser drilling on implantation rates following embryo transfer in day 3 synchronized pseudopregnant recipients was examined, the l laser drilled group (48.7%) was slightly higher than that of control group (43.6%). In addition, when the several pregnant mice delivered in two groups were analysed their chromosomal normality and tested reproductive ability, all p pups were presented normal chromosomal number (n=40) and showed normal growth and reproductive ability. Therefore, these results dem-onstrated that ZP drilling using a 1.48$\mu$m diode l laser can increase the embryo hatching and ind duce the normal pregnancy of mouse embryos.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.71-71
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• 2003

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine that has been implicated in the regulation of pre-implantation embryo development across several species. The aim of this study was to determine the effects of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) on development of porcine parthenotes and nuclear transferred embryos, and on their expression of implantation-related genes. In the presence of bovine serum albumin, mGM-CSF did not increase the percentage of oocytes that developed to the blastocyst stage and at day 7 did not increase oocyte cell number. Addition of 10 mM GM-CSF to protein-free culture medium significantly increased the compaction and blastocoel formation of 1- to 2-cell parthenotes and cloned embryos developing in vitro. However, cell number was not increased when they were cultured in the presence of GM-CSF. Semi-quantitative reverse transcripts polymerase chain reaction (RT-PCR) revealed that mGM-CSF enhances mRNA expression of the leukemia inhibitory factor receptor, but does not influence interleukin-6 or sodium/glucose co-transporter protein gene expression in blastocyst stage parthenotes. These results suggest that mGM-CSF may enhance viability of porcine embryos developing in vitro in a defined culture medium.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.21-27
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• 2020

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.

• Development and Reproduction
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• v.7 no.2
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• pp.95-103
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• 2003

Intracellular calcium is an important physiological factor in most cells, and ruthenium red and ryanodine play an important role as calcium modulators. Ruthenium red inhibits calcium-induced calcium release(CICR) from the intracellular calcium store. Ryanodine activates calcium release through ryanodine channel. The present experiment was performed to investigate the effects of two modulators on calcium ion metabolism and to determine their dose-dependency in oocyte and early embryo of mouse. Intracellular calcium ion concentration was measured in realtime by using confocal laser scanning microscope(CLSM) after loading of Fluo-3/AM in mouse oocytes and early embryos. Ruthenium red decreased intracellular calcium ion concentration in oocytes and early embryos at its high concentration(30, 300 $\mu$M). Ryanodine increased intracellular calcium ion concentration in oocytes and early embryos in low concentration(0.01 $\mu$M) but decreased that at higher concentrations(1, 10 $\mu$M). These results indicate that two modulators affected calcium ion metabolism in oocyte and early embryo of mouse, and their dose-dependency was different from somatic cell including myocytes.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• Proceedings of the Ginseng society Conference
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• 1978.09a
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• pp.43-49
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• 1978

This study was devised to observe the cytotoxic activity of extracts of Panax ginseng root against some cancer cells and to purify the crude extract. Three kinds of cancer cells(leukemic cells L5178Y, HeLa cells and Sarcoma 180 cells) and mouse embryo cells (as normal cells) were used for this study. The ginseng roots were extracted with petroleum ether in soxhlet apparatus, and the crude extracts were purified by the silicic acid column chromatography and thin-layer chromatography methods. The results obtained are summarized as follows; 1. Eight to ten mg of the petroleum ether extract (crude extract) were obtained from 1 g of Panax ginseng root, and its activities per mg were about 1,000 units. 2. Doubling time of the L5178Y cells was increased to two fold by 24 hours incubation in culture medium containing about one ${\mu}g$ of extract per ml, and eight and ten folds higher concentration of ginseng extract were required for the Sarcoma 180 cells and HeLa cells, respectively, than for the leukemic cells(L5178Y) to inhibit the cellular growth to the same degree. 3. When the L5178Y cells were exposed to medium containing various concentration of the extract for 24 hours before initiation of the soft agar cloning procedure, about $99\%$ of the L5178Y cells were killed at concentration of 8 units per ml. 4. The growth rate of mouse embryo cell (as normal cell) was not affected by the culture with media containing various amounts (1.45 to 30.0 ${\mu}g/ml$) of the extract. 5. The crude extract could be purified about four times by silicic acid column chromatography using several solvent systems, and one spot of active compound could be obtained on the thin-layer chromatogram. 6. In the Swiss mice inoculated with Sarcoma 180 cells, a survival time of the experimental group (injection group of active compound) was extended more. 1.5 to 2.0 times than the control group's(no injection group).

• Korean Journal of Animal Reproduction
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• v.10 no.2
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• pp.175-181
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• 1986

These experiments were carried out to clarify the effects various kinds of cryoprotectants which were frequently used in freezing embryos of domestic animals on the survival of frozen-thawed mouse embryos. As cryoprotectant, glycerol, DMSO and methanol were used and the procedures of adding them in medium were practiced by one-step or six-step adding method. Morphologically normal mouse embryos developed to blastocyst by in vitro culture after freezing and thawing were transferred to pseudopregnant recipients by surgical procedures. The results obtained in these experiments were summarized as follows: 1. The survival rates of the frozen-thawed 8-cell embryos, morulas and blastocysts following one-step addition of glycerol were 83.6, 80.3 adn 70.3%, respectively, while following six-step addition of glycerol, 69.2, 56.3 and 66.7% respectively. 2. When glycerol, DMSO and methanol were used as cryoprotectant under the same condition of freezing and thawing, the survival rates of frozen-thawed embryos were 74.0, 76.1 and 37.6%, respectively. 3. The implantation rate of embryos transferred to pseudopregnant recipients after freezing and thawing was 49.2%.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.66-66
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• 2003

The aim of this study was to investigate effect of different energy substrates on embryonic development of mouse embryos. Two cell embryos, recovered from ICR female mice (4 weeks old) at 44~52hrs after hCG injection (mated just after hCG injection), were cultured fur 72 hrs in the medium (MEM) supplemented with the three different energy substrates [glucose(G), pyruvate(P) and lactate(L)] and combinations (Control: 0 mM: group A: G 0.5; B: G 3.15; C: P 0.1; D: P 0.32; E: L 5.87; F: L 10.5; G: G0.5+P0.32+L10.5; H: G3.15+P0.1+L5.87; I: G0.5+P0.1+L5.87; J: G3.15+P0.32+L10.5). Blastocysts were stained differentially using PI and bisbenzimide. The 69.8% of the 2 cell embryos cultured in group F were developed the blastocysts. This was the highest (NS) than all other tested groups (44.2~62.8%). Blastocysts, cultured in the group E (60.4$\pm$26.9) and G (58.1$\pm$26.3), had significantly(p<0.05: group E vs. control, B, C, D; G vs. control, A, B, C, D) higher mean cell number compared with the other (42.6$\pm$25.8 ~ 55.2$\pm$31.3) and control (42.6$\pm$25.8) was at the basal level. The proportion of ICM (% ICM of total cells) in blastocysts cultured in group B (26.0$\pm$9.5%), C (29.6$\pm$22.8%) and J (26.0$\pm$11.8%) were significantly higher (p<0.05: control vs. group B, C, J: A vs. C, J; C vs. D, E, I) than those of other tested groups (15.0$\pm$10.6 ~ 23.8$\pm$ 12.9％) and control (15.0$\pm$10.6％) was at the basal level. These results showed that energy substrates supported the development of mouse 2 cell embryos, especially with greater embryo development in high dose of lactate added to media.