OBJECTIVE: To examine the effects of coculture with human oviductal cells regarding the development of 1-cell stage ICR mouse embryos and to investigate the effects of duration and start time of coculture. MATERIALS AND METHODS: ICR mice were superovulated with PMSG/hCG and 1-cell stage mouse embryos were recruited. 1-cell mouse embryos were cocultured on human oviductal cells in a CO2 incubator(coculture group) and were cultured on 0.4 % BSA+HTF media(control group)(Experiment 1). 1-cell mouse embryos were cocultured on human oviductal cells for 36, 44, 52, 60 hours after hCG IP respectively, and then were transferred to 0.4 % BSA+HTF media(Experiment 2). In comparison, 1-cell mouse embryos were cultured by using 0.4 % BSA+HTF media, and then were transferred to human oviductal cell coculture system using the same schedule(Experiment 3). Afterward, they were examined regarding the development to 2-cell, 4~8 cell stage mouse embryos, morulas and blastocysts. RESULTS: In experiment 1, the developmental rates to 2-cell embryos of coculture group and control group were 97.3 % and 98.7 %, respectively. After 2-cell embryos, coculture group showed significantly higher developmental rate than control group (p<0.05). In experiment 2, the developmental rates after 2-cell embryos showed the significant differences. The groups with coculture effects removed before post-hCG 60 hours showed significantly lower developmental rates (p<0.05). In experiment 3, the developmental rates after 2-cell embryos were higher when the coculture started at an earlier stage. Furthermore, the groups which were cocultured from post-hCG 52 hours exhibited significant lower developmental rate than the groups which were cocultured continuously (p<0.05). CONCLUSION: The coculture with human oviductal cell could improve the development of the embryos in vitro and might mimic the natural physiological condition better than media environment. The degree of improvement was more pronounced when the coculture started at an earlier stage and the duration of coculture was longer. More importantly, the changes of culture condition at post-hCG 52 hours in which secondary mitosis occurs, have significant detrimental effects on growth and development of mouse embryos.
Animals ; Blastocyst ; Coculture Techniques* ; Embryonic Structures* ; Growth and Development ; Humans* ; Mice ; Mice, Inbred ICR* ; Mitosis ; Morula ; Oviducts*

OBJECTIVE: To evaluat the effects of a culture medium with glucose in the presence of glutamine on the development of mouse embryos. METHODS: Two-cell embryos recovered from ICR mice at 48 hrs after hCG injection (mated just after hCG injection) were cultured in DMEM (with 20% hFF) supplemented with or without glucose on the presence of glutamine. Embryos were cultured under three different glucose regimens: (1) 0 mM (control); (2) 0.5 mM (group I); or (3) 3.15 mM (group II), and were analyzed at 24, 48, 72 and 96 hours intervals. Chi-square test (x2-test) was used to compare values of groups. RESULTS: No differences were found in the number of embryos showing morula (control: 37.5%; group I: 51.0%; group II: 48.4%), blastocyst (control: 21.5%; group I: 33.3%; group II: 34.4%) and blastocyst and hatching or hatched blastocyst (control: 81.9%; group I: 83.3%; group II: 82.8%) between groups at 24 hrs, 48 hrs or 72 hrs respectively. However at 96 hrs, the number of hatched and attached blastocyst was significantly higher in group I (82.3%) and II (78.5%) than control (63.2%; P<0.05). CONCLUSION: The addition of glucose (0.5 mM) to the DMEM, as energy source, improved the rate of development of late stage embryos in mice.
Animals ; Blastocyst ; Eagles* ; Embryonic Development* ; Embryonic Structures* ; Female ; Glucose* ; Glutamine* ; Mice* ; Mice, Inbred ICR ; Morula ; Pregnancy

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.
Animals ; Blastocyst ; Embryonic Structures* ; Ethylene Glycol ; Ficoll ; Humans ; Mice* ; Morula ; Sucrose ; Survival Rate ; Vitrification*

The present study was designed to define the role of prostaglandin in the development and hatching of mouse embryo. The effects of indomethacin, an inhibitor of prostaglandin synthesis, on the development and hatching of morula and blastocyst were examined. In early morula stage, embryos were degenerated significantly at 100 muM and 200 muM indomethacin. However, :he viability of embryos was not influenced by concentration in any other embryonic stages. In all embryonic stages, the hatching was suppressed with concentration dependent manner, but expansion was not suppressed. Particularly, in 84h embryos post hCG injection, the hatching was suppressed significantly compared with post hCG 72h or 96h embryos. When embryos were treated with 100 muM indomethacin for a specific time (12h) in according to the development stage, the hatching was suppressed all groups. These suppressional effect was decreased as embryonic development stage was progressed. However, the expansion was not affected in all treatment group. This study suggests that hatching-related metabolic substances are synthesized from morula stage and intraembryonic signaling mediated prostaglandin was important for development and hatching of mouse embryo.
Animals ; Blastocyst ; Embryonic Development ; Embryonic Structures* ; Female ; Indomethacin* ; Mice* ; Morula ; Pregnancy

OBJECTIVE: This study aimed to examine the effect of laser-assisted zona thinning (LAZT) at one or four-points on the blastocyst formation and hatching process in mice with respect to female age. METHODS: Eight-cell or morula embryos collected from superovulated C57BL female mice with different ages (6-11 and 28-31 weeks) were treated with LAZT at one-point (LAZT1) or four-points (LAZT4). The zona pellucida was thinned to more than 70% of its initial thickness by making two holes of 15-20 microm. RESULTS: In the young mice, LAZT resulted in a significant increase in early hatching and hatching rates compared to the control group (p<0.05). However, in the old mice, LAZT significantly increased blastocyst formation as well as early hatching and hatching compared to the controls (p<0.05). These effects were more remarkable in LAZT4 than in LAZT1 and in aged mice than in young ones. CONCLUSION: These results show that multi-point LAZT leads to a significant improvement of blastocyst formation and hatching in mice compared to controls.
Animals ; Blastocyst ; Embryonic Structures* ; Female ; Herpes Zoster* ; Humans ; Mice* ; Morula ; Zona Pellucida

OBJECTIVE: The objective of this study was to examine the effect on development of mouse preimplantation embryos in culture media with different composition of energy sources in vitro culture. METHODS: Two hundred and seventy one two-cell embryos were cultured in four different culture system for 96 hours. Group I (n=61) was cultured in DMEM-G (DMEM with glutamine) only, groupII (n=64) was cultured in DMEM-GGP (DMEM with glutamine, glucose and pyruvate) only, group III (n=72) was cultured for 48 hours in DMEM-G and then transferred to DMEM-GGP and group IV (n=74) was cultured for 48 hours in DMEM-GGP and then transferred to DMEM-G. Development of embryos in each group was observed every 24 hours. RESULTS: After 24 hours, the rate of development > or = 3-cell was significantly higher in groupII (87.5%) and IV (86.5%) compared with group I (59.0%) and III (62.5%). After 48 hours, the rate of development into > or = morula stage was significantly higher in GroupII (79.7%) and IV (86.5%) compared with group I (34.4%) and III (37.5%). After 72 hours, the rate of development into blastocyst was significantly higher in group IV (74.3%) compared with group I (49.2%) and III (45.8%). After 96 hours, the rate of development into > or = expanded blastocyst was significantly higher in group IV (70.3%) compared with group I (32.8%),II (53.1%), and group III (40.3%). CONCLUSION: Mouse preimplantation embryos development was the most effective in culture system with DMEM-GGP for 48 hours and then transferred to DMEM-G.
Animals ; Blastocyst* ; Culture Media* ; Embryonic Structures ; Glucose ; Glutamine ; Mice* ; Morula ; Pyruvic Acid

OBJECTIVE: To find out the optimal freezing time in terms of developmental stage of mouse embryo and the effectiveness of vitrification method for freezing them. METHODS: Superovulation induction was performed using PMSG and hCG. Total 1,437 mouse embryos (vitrification group: 743, slow-freezing group: 694) were obtained and cultured with the T6 containing 0.4% BSA medium. Each developmental stage of embryos (1-, 2-, 4-, 8-cell, morula and blastocyst) were cryopreserved by vitrification and also by slow freezing method for comparison of the results. After thawing, the recovery rate, the survival rate and the blastocyst developmental rate were analysed and compared in two different settings. Student's t-test was used for statistical analysis. RESULTS: The survival and developmental rates at all subgroups of vitrification method are significantly higher than those of slow-freezing groups, but not in the recovery rates. In vitrification group, the survival rate and the blastocyst developmental rate are highest when frozen at morula stage, 98.4% and 86.4%, respectively. In slow-freezing group, the survival rate is also highest when frozen at morula stage, 87.2%, and the blastocyst developmental rate is highest when frozen at 8-cell stage, 78.1%. CONCLUSION: The vitrification method is more efficient for mouse embryo freezing compared with slow freezing one. Among various developmental stages of mouse embryos, morula stage seems to be the optimal stage for cryopreservation, whatever the freezing method applied. Therefore, we recommend embryo freezing at morula stage by vitrification method.
Animals ; Blastocyst ; Cryopreservation ; Embryonic Structures* ; Freezing ; Mice* ; Morula ; Superovulation ; Survival Rate ; Vitrification*

OBJECTIVES: The aim of this study was to evaluate the influence of three different media on preimplatation embryo development and the expression of Bcl-2, Mcl-1, Bax, and Bok in mouse. MATERiALS AND METHODS: Two-cell embryos were retrieved from iCR female mice (4 weeks old) at 48 hr after hCG injection and cultured in Ham's F-10, HTF, and G1.2 media. The developmental rate of 2-cell embryos was evaluated from 24 hr to 72 hr after culture. RT-PCR was performed for the detection of Bcl-2, Mcl-1, Bax, and Bok gene expression. RESULTS: The rates of morula and blastocyst in HTF and G1.2 media (88%, 98.1%) were significantly higher than those in Ham's F-10 media (39.6%) at 48 hr. Likewise, the rates of hatching and hatched blastocyst in HTF and G1.2 media (21.9%, 52.9%) were higher than those in Ham's F-10 media (3.5%) at 72 hr. Bcl-2 and Bax mRNAs were highly detected in embryos cultured in Ham's F-10 when compared in embryos cultured in HTF and G1.2. in contrast, the expression of Mcl-1 and Bok was not significantly different. CONCLUSION: These results show that HTF and G1.2 culture media increase the rate of blastocyst formation and stimulate Bcl-2 and Bax gene expression in mouse preimplantation embryos.
Animals ; Blastocyst* ; Culture Media ; Embryonic Development* ; Embryonic Structures ; Female ; Gene Expression ; Humans ; Mice* ; Morula ; Pregnancy ; RNA, Messenger

OBJECTIVE: To ananlyze the direct effect of nitric oxide (NO), generated from sodium prusside (SNP) on the embryo developments in reproductive process. DESIGN: ova from mouse were treated to allow fertilization in in vitro culture. And the samples of fertilized ova were alloted into five alliqutos. Each alliquot was cultured in media treated with either concentration at 0 (n=92), 25microM (n=84), 50microM (n=80), 100microM (n=77), 500microM (n=54) of SNP. Main Outcome MEASURE: Rates of embryonal cell cleavages, viability and cell morphology were assessed during in vitro fertilization and culture. RESULTS: As analyse the cell cleavage at 24 hours after in vitro culture of fertilised egg in variuos NO concentration, all of egg cells of each alliquot were developed to 2~4 cell stage. But the alliquot of egg cells treated with 500microM, which were totally degenerated. And also all embryonal cells of each alliquot were developed to 8 cell stage and morula stage on culture continuosly. And the embryonal cells of each alliquot were analysed at 24 and 48 hours following the in vitro culture. The rates of cell fragmentation and fusion were 4.2+/-3.4% in control group which is not treated with NO, while experimental groups was high, as rated 23.4+/-6.2% in 25microM, 28.2+/-5.7% in 50microM and 32.1+/-6.4% in 100microM concentration of NO. Accordingly the rate of abnormal morphology of embryonal cell in control was lower significantly than that in each alliquot of experimental groups (p<0.05). And the degenerated rates of embryonal cells were 0% in control, 17.8+/-6.7% in 25microM, 23.6+/-4.7% in 50microM and 26.8+/-11.2% in 100microM at 8 cells and morula on culture of 48 and 72 hours. On the examination of embryonal cells developed to blastocyst through in vitro culture, the rates of degenerated cells were 16.8+/-7.2% in control, 37.5+/-6.2% in 25microM, 73.4+/-4.6% in 50microM, 100% in 100microM. CONCLUSION: This results suggeted that the No in any concentrations is harmful on embryos in view of morphology as well as viability of cell, and the toxicity of No on embryo is stronger at condition in higher concentration of NO.
Animals ; Blastocyst ; Embryonic Structures ; Fertilization ; Fertilization in Vitro ; Mice* ; Morula ; Nitric Oxide* ; Outcome Assessment (Health Care) ; Ovum ; Sodium

OBJECTIVE: The purpose of this present study was to compare mouse embryo development in 3 commercial media and hatching competence of mouse embryo with or without enzymatic treatment. METHODS: Collected 375 mouse embryos were divided into three groups, and then cultured in IVF-20 (G2), Medicult IVF (M3), P-1 (blastocyst M), respectively. Three day mouse morulae were cultured in G2 media treated with pronase. The results were analyzed using Chi-square test, and considered statistically significant when p<0.01. RESULTS: The developmental rate of 2 cell mouse embryo after 72 hours was highest in IVF-20 (G2) among conventional 3 media. The hatching rate of mouse morulae was low when clultured in G2 media without pronase during 48 hours. However, it was higher when cultured in media treated with l mg/ml, 2.5 mg/ml, 5 mg/ml pronase, respectively. CONCLUSIONS: Using good media and digestion of zona pellucida with enzymatic treatment improve development and hatching rate of embryo. Therefore, implantation and pregnancy rate could be improved.
Animals ; Digestion ; Embryonic Development ; Embryonic Structures* ; Female ; Mental Competency ; Mice* ; Morula ; Pregnancy ; Pregnancy Rate ; Pronase* ; Zona Pellucida