• Journal of Embryo Transfer
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• v.4 no.1
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• pp.28-34
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• 1989

These experiments were carried out to determine the effect of cell stage in embryo bisection on the sub-Sequent in vitro and in vivo development in mouse. The embryos of ICR mouse were microsurgicaily bisected at 2-cell, 4-cell, 8-cell, morula and blastocyst stage using a microsurgical blade attached a micromanipulator. These demi-embryos without zona pellucida were cultured up to blastocyst stage and transferred to pseudopregnant mice, and the development of these demi-embryos was compared with the results of intact embryos of the corresponding cell stage. The successful rate of mouse embryo bisection at 4-cell stage (59.0%) was significantly (p <0.05) lower than those at 8-cell (75.6%), 2ce11 (80.7%) or morula stage (84.8%), and highest at blastocyst stage (95.7%). When the bisected embryos without any damage from microsurgery were cultured in vitro up to blastocyst,the in vitro de'velopment of demi-embroys bisected at morula to blastocyst was 91.6 to 95.3%, which was similar to the culture result of intact embryos of corresponding stage. However, the in vitro development of demi-em-bryos bisected at 2- to 8-cell stage was signiflcantiy (p <0.05) lower.The post-transfer implantation rate of demi-embryos developed in vitro to eu-blastocyst were 19.6 and 25.4% in demi-embryos bisected at morula and blastocyst stage,respectively and not significantly (P <0.05)different from the result of intact embryos of the same stage. However, the implantation rates of demi-embryos bisected at 2- or 8-cell stage were significantly (P <0.05) lower than the result from the intact embryos of the corresponding stage.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.177-186
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• 1998

While tubal pregnancy is frequently observed in human, it has been reported to rarely occur in other mammals. To investigate the reason of the absence of tubal pregnancy in other mammals, the ability of bovine tubal(oviductal) fluid to su, pp.rt the outgrowth of mouse embryos waw examined by using an in vitro model system wherein the trophoblast cells of hatched mouse blastocysts attach to and outgrow on tissue culture plates coated with FBS. When mouse blastocysts grwon in vitro from 2-cell embryos were cultrued in the dishes coated with FBS, human follicular fluid(hFF) and bovine follicular fluid(bFF), respectively, underwent outgrowth by spreading onto the plastic dishes during 48 hr. In contrast, none of the embryos cultured in the dishes coated with BSA or bovine obiductal fluid(bOF) did outgrow but remained as late blastocysts. Since addition of bOF at 5mg/ml or higher conc. to the culture medium resulted in degeneration of all embryos during 48 hr culture, 10mM conc. of glutathione(GSH) was added to the bOF-containing medium to circumvent the toxicity of bOF. In addition, bOF was heated $65^{\circ}C$ for 30 min(hbOF) to get rid of its precipitating properties and then added to the culture medium. When blastocysts were cultured in the presence of both hbOF and GSH 45.4% of embryos attached to the culture dishes. However, none of these embryos underwent outgrowth. Fially embryos were cultured in the presence of both hbOF and GSH but in the dishes coated with FBS. When they were examined after 48 hr, all of the blastocysts exhibited well-developed outgrwoth. Based upon these results, it is concluded that bovine oviductal fluid is capable of su, pp.rting the attchment of mouse blastocysts onto the culture plaste whereas it cannot promote the outgrwoth of mouse blastocysts in vitro, probably due to the lack of outgrwoth factor.

• Journal of Embryo Transfer
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• v.9 no.2
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• pp.173-180
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• 1994

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen and thawed rapidly. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

• Korean Journal of Animal Reproduction
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• v.8 no.2
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• pp.116-121
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• 1984

These experiments were carried out to examine the effect of rapid thawing (500$^{\circ}C$/min) on the survival of 8-cell mouse embryos cooled slowly (0.5-1$^{\circ}C$/min) to precooling temperatures between -10 and 070$^{\circ}C$ before direct transfer ofembryos to -196$^{\circ}C$, and to compare the survival of embroys thawed slowly (20$^{\circ}C$/min) and rapidly (500$^{\circ}C$/min) after in vitro culture. In addition, the sensitivity of 8-cell mouse embroys to the rate of addition and removal of cryoprotectant at room temperauture, and the effect of developing stages on the survival of embryos frozen-thawed slowly were investigated. The results obtained were summarized as follows: 1. Embryos were survived in rapid thawing (500$^{\circ}C$/min) only when slow cooling was terminated at relatively high subzero teperaure (-20 to -60$^{\circ}C$). The highest survival rate(77.0%) in in vitro culture of embryos thawed rapidly was obtaeined after transfer to -196$^{\circ}C$ from -40$^{\circ}C$. 2. For the survival of embryos in slow thawing (20$^{\circ}C$/min.), slow cooling to lower subzero temperature (-50$^{\circ}C$ and below) was required before transfer of embryos to -196$^{\circ}C$. These results indicate that embryos transferred to -196$^{\circ}C$ from high subzero temprature contain much interacellular ice to damage them during slow warming but to permit survival of embryos after rapid warming. 3. The Survival rate (72.7%) of 8-cell mouse embryos after rapid addition and removal of cryoprotectant, DMSO at room temperature was similar to that (83.9%) after slow addition and removal of cryoprotectant at same temperature. 4. The survival rates of 1-, 2-, 4- and 8-cell embryos frozen-thawed slowly were 26.7, 76.4, 70.0 and 83.9%, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.3
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• pp.119-125
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• 2017

Objective: In vitro culture of preimplantation embryos is improved by grouping embryos together in a drop of media. Individually cultured embryos are deprived of paracrine factors; with this in mind, we investigated whether the addition of a single embryo-secreted factor, interleukin-6 (IL-6), could improve the development of individually cultured embryos. Methods: Mouse embryos were cultured individually in $2{\mu}L$ of G1/G2 media in 5% oxygen and supplemented with a range of doses of recombinant mouse or human IL-6. Results: Mouse IL-6 increased hatching at doses of 0.01 and 10 ng/mL compared to the control (93% and 93% vs. 78%, p< 0.05) and increased the total number of cells at a dose of 0.1 ng/mL compared to the control ($101.95{\pm}3.36$ vs. $91.31{\pm}3.33$, p< 0.05). In contrast, the highest dose of 100 ng/mL reduced the total number of cells ($79.86{\pm}3.29$, p< 0.05). Supplementation with human IL-6 had a different effect, with no change in hatching or total cell numbers, but an increase in the percentage of inner cell mass per embryo at doses of 0.1, 1, and 100 ng/mL compared to the control ($22.9%{\pm}1.1%$, $23.3%{\pm}1.1%$, and $23.1%{\pm}1.1%$ vs. $19.5%{\pm}1.0%$, p< 0.05). Conclusion: These data show that IL-6 improved mouse embryo development when cultured individually in complex media; however, an excess of IL-6 may be detrimental. Additionally, these data indicate that there is some cross-species benefit of human IL-6 for mouse embryos, but possibly through a different mechanism than for mouse IL-6.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.283-289
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• 2000

Objective: The study was performed to compare the survival rate and the development of day 2 mouse embryos which had freezing procedures done. Methods: We used three different vitrification solutions (EFS, VS14, DPS) and a ultrarapid freezing solution (UFS) for cryopreservation of day 2 mouse embryo. Results: We tested toxicity by exposing embryos to vitirification solutions and a ultrarapid freezing solution. The survival rates are 100%, 97.8%, 95.6% and 100% (EFS, VS14, DPS and UFS). After cultured for 96 hours, hatching rates of each group are 93.5% (no freezing), 95.6% (EFS), 86.4% (VS14), 93.0% (DPS), and 93.0% (UFS). There is no significant differences among groups. The survival rates after thawing cryopreserved embryos are 80.2%, 91.7%, 69.5%, 0% and 91.8% (slow freezing, EFS, VS14, DPS and UFS). Also cultured for 96 hours, the hatching rates are 93.5% (no freezing), 84.1% (slow freezing), 93.9% (EFS), 48.5% (VS14) and 70.1% (UFS). Conclusion: The survival rates of vitrification in EFS solution and ultrarapid freezing are higher than slow freezing (p<0.05). The hatching rate of vitrification in EFS solution cultured for 96 hours is highest, so vitrification of day 2 mouse embryos in EFS solution considered as more effective for cryopreservation.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.71-81
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• 1994

These experiments were carried out to investigate the effects of human follicular fluid (hFF) as a protein supplement on development of mammalian embryo as well as to find out ways toward effective use of hFF. The developmental rates of mouse embryos to the blastocyst and implantation stages were significantly higher in T6 ＋hFF than T6＋hFCS. Classified hFF according to the maturity of contained oocytes (M-hFF and Im-hFF), and compared the rates of development of mouse embryo cultured in M-hFF or Im-hFF to culture medium T6. Total protein, albumin and estradiol concentrations were higher in M-hFF than Im-hFF (P<0.05). The developmental rates of mouse embryos to the blastocyst and hatching blastocyst stages cultured in Im-hFF were significantly lower than those in M-hFF and the basic medium. In accordance of the results of human IVF, hFF has been divided into 4 groups. The developmental rates of mouse embryos to the blastocyst stage in presense of hFF from pregnant patients, who have good grade embryos, were significantly higher than those in hFF from patients who have poor grade embryos or were not pregnant. In addition, the rates of development of human embryo were compared in presense of BSA, hFF or hFCS. The developmental rates of human embryos cultured in Ham's F10＋hFF were significantly higher than those in the Ham's F10＋BSA. These results suggests that the culture system using hFF could improve the development ability of mammalian embryos and the viability of blastocysts cultured in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.2
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• pp.167-172
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• 1990

This study was carried out to clarify the effects of various kinds of cryoprotectants which were frequently used in freezing embryos of domestic animals on the survival of frozen-thawed mouse embryos. Mouse embryos were collected by hyperstimulation induction of ICR mouse. The samples were slowly cooled ($l^{\circ}C/min$) to temperatures between $-7^{\circ}C$ and $-30^{circ}C$ before direct transfer to liquid nitrogen ($-196^{\circ}C$) and thawed rapidly ($-500^{\circ}C$/min). As cryoprotectants, Glycerol, DMSO, Ethylene glycol and Propylene glycol were used and applied each 2 cell, 8 cell, morula in embryo stage. After normal mouse embryos developed to blastocyst by in vitro culture, we observed recovery rate and developing rate of embryos at thawing. The results obtained in these experiments were as follows : 1. The in vitro development rate from the frozen-thawed 2 cell embryos to the blastocyst were 67.7% in ethylene glycol, 65.7% in Propylene glycol, 55.2% in glycerol and 50.0% in DMSO respectively. 2. The in vitro development rate from the frozen-thawed 8 cell embryos to the blastocyst were 83.6% in DMSO, 75.7% in glycerol, 52.2% in propylene glycol respectively. 3. The in vitro development rate from the frozen-thawed morula to the blastocyst were 84.2% in glycerol, 80.0% in DMSO, 66.6% in propylene glycol and 55.2% in ethylene glycol respectively.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.30-30
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• 2003

The present study examined the developmental ability of embryonic stem (ES) cells aggregated with mouse parthenogenetic embryos. Oocytes obtained from superovulated female mouse (BCF1) were treated with 7% ethanol and 5 $\mu\textrm{g}$/$m\ell$ cytochalasin B (CB) for producing pathenotes and in vitro fertilized with fresh sperm for producing normal embryos. The reporter vector (pNeoEGFP) were inserted into ES cells (129S4/svJae) by electroporation. At the 8-cell stage, in vitro fertilized embryos and pathenotes, which the zona pellucida was removed, were co-cultured with 5~10 ES cells for 4 hr. After in vitro fertilized embryos and parthenotes aggregated with ES cells were incubated to blastocyst stage, and these blastocysts transferred into the uterus of pseudopregnant recipients. The fertilized embryos aggregated with ES cells were successfully developed to offspring, but the parthenotes aggregated with ES cells failed to develop offsprings. However, genomic DNA of ES cells was detected in the pathenogenetic fetus by polymerase chain reactions at 15 day post gestation. In this study, results indicated that parthenotes aggregated with ES cells showed possible development to fetus. In the future, this method may help to produce transgenic chimera from parthenotes aggregated with ES cells.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.1-4
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• 2008

This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze $2{\sim}4$ cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in $LN_2$. Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for $96{\sim}120\;hr$ and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.