• Journal of Embryo Transfer
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• v.8 no.1
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• pp.13-19
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• 1993

These experiments were carried out to determine the effect of pregnancy in bisected embryo. The embryos of ICR mouse were microsurgically bisected at morula and blastocyst stage using microsurgical blade attached a micromanipulator. These bisected embryos without zona pellucida were cultured up to blastocyst stage and cell count and diameter of stained blastomere, and transferred pseudopregnant mice. And the development of these bisected embryos was compared with the results of production of young of the corresponding intact embryos or cell stage. When the bisected mouse embryos were cultured in vitro for 20 to 24 hours in morula stage(77.2%) or 3 to 6 hours in blastocyst stage(84.1%), them were developed to the expanded blastocyst stage. There were no significant(P<0.05) differences in the development rate of bisected embryos between in morula and blastocyst stages. The embryo size of blastocyst developed in vitro from bisected embryo was small(P<0.05)than intact embryo. However, the number of blastomeres with bisected embryo (24.7+1.3and 21.5+1.2 respectively) were significantly(P<0.05) reduced, compared with that of intacted embryos(36.3+1.1 and 41.4+1.2 respectively). When compared with the result of pregnancy rate(63.6%) after surgical transfer of bisected morulae, a similar result(65.4%) was obtained with bisected blastocyst stage(P< 0.05). However, production of youngs (38.8%) after transfer of bisected morula, a similar result (38.1%) was obtained with bisected blastocyst stage (P<0.05).

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.81-88
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• 1995

Culture medium (ASF-301) of tHUE-2 cell, human endothelial cell line, and culture medium of these cells (conditioned medium : CM) which affect embryonic development of in vivo fertilized 1-cell embryos of mouse were examined. Two-cell stage block of mouse embryos was overicomed in ASF-301 and CM without EDTA, which usually added in basic medium (modified Whitten Medium: MWM, control) to overcome the 2-cell stage block. The developmental rates of embryos to the blastocyst stage were significantly increased in MWM containing 12.5% of growth factors added to ASF-301 (10mg/ $\ell$ transferrin, 1mg/$\ell$ insulin, 0.01mg/$\ell$ EGF) than those of 100% addition and control, 78.0% vs 20.8 and 52.3%, respectively (P<0.05), but the growth factors was not affected the hatching rate of blastocyst. Using ASF-301 or CM which was not treated, embryonic development into the blastocyst and hatched blastocyst stages were not affected. However, proportions of embryonic development into the blastocyst and hatched blastocyst stages were significantly higher in dilution (ASF-301 1:10; CM 1:3~1:6) than those in control (P,0.05). In ASF-301 dialyzed M.W.<10000 dialysis membrane, the developmental rate upto the hatched blastocyst stage was significantly increased, compared to ASF-301 which was not dialyzed (P<0.05), and hatching rate of blastocyst of these group was singnificantly increased than those in MWM (P<0.05). Compared to CM which was not dialyzed, however, in dialyzed CM was significantly decreased, compared to untreated CM (P<0.05), especially any hatched blastocyst was not appeared. As a result of these experiments indicated that a kind or porper treatment such as a dilution of complex synthetic cell culture medium and conditioned medium, and that a optimal concentration of growth factors are usuful for embryo cultrue in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.1
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• pp.9-17
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• 1993

This study was carried out investigate the effect of water quality and the kind of media on the in vitro development of 1-cell and stage mouse embryos. $F_1$ hybrid mice were superovulated and timely mated. 1-cell stage and 2-cell stage mouse embryos were recruited and taken into Ham's F-10 or m-KRB media which was made of two of two kinds of water having different quality, highly purified water and tap water. 2-cell stage embryos grew up well in vitro to blastocyst or hatching blastocyst regardless of the composition of culture media, but 1-cell stage mouse embryo didn't develop well to blastocyst or hatching blastocyst in simple media like m-KRB. These results meant in vitro devleopment of 1-cell stage mouse embryo neded complex media like Ham's F-10 which contained abundant protein components. In case of quality control for water, in vitro fertilization program. observation of in vitro development of 2-cell mouse embryos up to blastocyst or hatching blastocyst media such as m-KRB would be efficatious in detecting the difference of water quality.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.2
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• pp.177-185
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• 2005

Objective: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. Materials and Methods: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. Results: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. Conclusion: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

• 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.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.63-69
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• 2012

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ($p$ <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ($p$ <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.247-251
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• 1997

This research was conducted to observe developmental capacity of the early embryos aggrigated to phytohemagglutinin-M(PHA-M) in the culture of mouse embryos in vitro. The results showed that the development of blastocyst increased to 2-celT >< 2-cell : 68. 9%, 4-cell $\times$4-cell : 92.5% and 8-cell $\times$8-cell : 97.3% in the aggrigated embryos of ICR mouse, and 2-cell $\times$ 2-cell : 90.0%, 4-cell $\times$4-cell : 93.9% and 8-cell $\times$ 8-cell : 100% in the aggrigated embryos of two different strains (ICR $\times$ CBA/J mouse). (Key words : aggrigated embryos, in vitro 2-cell block, phytohemagglutinin-M, blastocyst)

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.307-313
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• 2021

Traf4 (Tumor necrosis factor Receptor Associated Factor 4) is a member of the tumor necrosis factor receptor (TNFR) - associated factors (TRAFs) family. TRAF4 is overexpressed in tumor cells such as breast cancer and associated with cytoskeleton and membrane fraction. Interestingly, TRAF4 was localized with tight junctions (TJs) proteins including OCLN and TJP1 in mammary epithelial cells. However, the expression patterns and biological function of Traf4 were not examined in preimplantation mouse embryos although Traf4-deficient mouse showed embryonic lethality or various dramatic malformation. In this study, we examined the temporal and spatial expression patterns of mouse Traf4 during preimplantation development by qRT-PCR and immunostaining, and its biological function by using siRNA injection. We found upregulation of Traf4 from the 8-cell stage onwards and apical region of cell - cell contact sites at morula and blastocyst embryos. Moreover, Traf4 knockdown led to defective TJs without alteration of genes associated with TJ assembly but elevated p21 expression at the KD morula. Taken together, Traf4 is required for TJs assembly and cell proliferation during morula to blastocyst transition.

• Journal of Animal Science and Technology
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• v.65 no.4
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• pp.767-778
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• 2023

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.2
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• pp.174-180
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• 2009

This study was carried out to establish an appropriate condition for the efficient cryopreservation of the mouse pronuclear embryo. In vitro cryopreservation of pronuclear embryos was carried out by slow freezing or vitrification methods and development rate of 2-cell, blastocyst and hatched blastocyst was measured as well as survival rate of the thawed pronuclear embryo. After slow freezing, vitrification and thawing of mouse pronuclear embryos, the survival rate and blastocyst development rate for the vitrification group was 97.3 and 53.4%, respectively, which was significantly higher as compared to the slow freezing group with 88.6 and 23.9%, respectively (p<0.05). Blastocyst developmental rate in each experimental group was significantly higher for 21 h in the post-hCG group at 40.5-57.0% than the 24 h post-hCG group at 40.5% (p<0.05). ICM (Inner cell mass) cell numbers of blastocyst-stage embryos during the different stages of mouse pronuclear embryos, slow freezing and vitrification period in the control and vitrification groups were 22.1${\pm}$2.7 and 17.0${\pm}$3.1-22.0${\pm}$3.2, respectively; hence, the slow freezing group (10.2${\pm}$2.0) had significantly higher cell numbers than those of the other two groups (p<0.05). Trophoblast (TE) cell number in the control group, 65.8${\pm}$12.6, was significantly higher than in the slow freezing group, 41.6${\pm}$11.1 (p<0.05). The total cell numbers in the control group and 21 h post hCG group were 87.9${\pm}$13.6 and 81.8${\pm}$14.1, respectively, and were significantly higher than for the slow freezing group (51.8${\pm}$12.6; p<0.05).