• Korean Journal of Animal Reproduction
• /
• v.11 no.2
• /
• pp.127-131
• /
• 1987

This study was carried out to obtain the basic information on splitting and culture of mouse and bovine embryos. Two-, four-, eight-, cell and morula mouse embryos were digested with pronase, splitted in vitro by micro-glass needel with hand, and bovine embryos were splitted by micromanipulator. The splitted embryos were cultured under 5% of CO2 gas in air at 37$^{\circ}C$ for 48-72 hours. The results obtained in this study were summarized as follows: 1. The mouse and cattle were superovulated by 5IU of PMS and HCG, and 2500IU of PMS and 25mg of PGF2$\alpha$, respectively. The average number of embryos after superovulation were 32.5$\pm$8.2 and 7.5$\pm$3:1, respectively. 2. Out of total 122 embryos splitted, the successful splitting rate was 75.0%, 66.7%, 68.4% and 71.4% in 2-, 4-, 8- and morula embryos in mouse, respectively. There was no different splitting rate between mouse(71.4%) and bovine embryos(66.7%) in morula. 3. The successful culture rate of splitted embryos was 68.0% and 67.9% in mouse and bovine embryos, respectively.

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

• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.31-31
• /
• 2001

This study was to establish in uitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, This study was to establish in vitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, $\geq$morula: 4.8%) and 7 hrs ($\geq$2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.morula: 4.8%) and 7 hrs (2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.

• Journal of Embryo Transfer
• /
• v.8 no.1
• /
• pp.13-19
• /
• 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
• /
• v.12 no.1
• /
• pp.37-41
• /
• 1988

These studies were carried out to examine the sex of preimplantation mouse embryo. For the investigation of sex-ration of mouse embryos, morula and blastocysts stage embryos treated with H-Y antiserum (10%, v/v) and FITC anti-mouse-IgG were divided into the positive and negative embryos. Positive and negative identified embryos were observed the viability according to the in vitro cultured and the sex ratio was also investigated by chromosomal analysis. The results obtained in these studies were summarized as follows: 1. Two hundred sixty-seven recovered embryos of morula or blastocyst stage were incubated in medium containing H-Y antiserum and FITC anti-mouse-IgG. Positively or negatively identified embryos were 139 and 128. This trend indicated the approximal sex ratio was 1:1. 2. Sex ratio was measured using the embryos treated with indirect immunofluorescence assay to examine the relationship between embryo developmental stage and sex ratio. Sex ratio of morula stage embryos was 45.2% positive and 54.8% negative, on the other hand, the ratio switched to 56.4% positive and 43.6% negative embryo in blastocyst stage. 3. Fourty-seven positive and 57 negative embryos were obtained out of 104 morula stage embryos treated with indirect immunofluorescence assay. Survived positive or negative embryos during in vitro culture were 42 and 49, respectively out of 47 and 57 embryos. 4. The numbers of negative and positive embryos were 171 and 92 out of 163 blastocyst embryos which were incubated in the medium containing H-Y antiserum and FITC anti-mouse-IgG. The result of karyotype test showed the successful rate of sexing embryo is positive and negative embryos was63.0% (58/92) and 62.0% (44/71). The final female to male ratio within 58 positive embryos was 22.7:77.6, and the ratio of the 44 negative embryos was 77.3:22.7.

• Clinical and Experimental Reproductive Medicine
• /
• v.24 no.1
• /
• pp.35-42
• /
• 1997

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 ${\mu}M$ and 200 ${\mu}M$ indomethacin. However, the 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 ${\mu}M$ 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.

• Clinical and Experimental Reproductive Medicine
• /
• v.19 no.2
• /
• pp.125-131
• /
• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• Korean Journal of Animal Reproduction
• /
• v.11 no.3
• /
• pp.218-222
• /
• 1987

This study was conducted using inbred ICR mice to investigate the sex-ratio of preimplantation mouse embryos. For the investigation of sex-ratio of mouse embryos, the karyotype of embryos collected at 70-72, 74-76, 78-80 and 82-84 hr after HCG injection was analyzed by chromosomal analysis. Eight-cell embryos were cultrued up to blastocyst stage, then divided them into three groups(fast-, intermediate- and slow-) according to the blastocoel formation. The sex-ratio was also investigated by chromosomal analysis. 1. The highest apperance of eight-cell and morula was observed at the embryos collected respectively at 66-68 hr(84.6%) and 82-84 hr(79.3%) compared to any other group. 2. The successful rate of embryos sexing at 4-, 8-cell and morula stage were 23.1% (3/13), 42.1%(138/328) and 32.6%(47/141), respectively. The respective sex ratios (female vs male) of 4-, 8-cell and morula were 66.7:33.3, 49.3:50.7 and 39.5:60.5. 3. Of the 476 eight-cell embryos cultured in vitro, 427(89.7%) embryos were developed to the blastocysts and the number of fast-, intermediate- and show-developing embryos were 139, 144 and 144, respectively. 4. Female to male ratios fo fast-, intermediate- and slow-developing group were 23.0:77.0, 55.2:44.8 and 73.8:26.2, respectively. Significantly higher (P<0.05) number of female (48/65;73.8%) was observed in the group of slow-developing embryo than that out of total number of embryos(82/188;43.6%).

• Korean Journal of Animal Reproduction
• /
• v.16 no.3
• /
• pp.217-224
• /
• 1992

These studies were carried out ot investigate on the transferred embryo development following ultrarapid frozen for 8-cell and morula of in vitro fertilization mouse embryos. The post-thaw embryo survival was evaluated and compared by cell stage of embryos and by equilibration time before ultrarapid freezing. The results obatined were summerized as follows: 1. The effects of equilibration time of 3 vs. 6 minutes before ultrarapid freezing and after thawing on the morphological survival and the viability of 8-cell and morulas embryos were not significant. 2. When the ultrarapid frozen-thawed 32 eight-cell and 33 morula embryos, and 30 fresh blastocysts were transferred to pseudopregnant recipient mice, the number of normal offsprings produced were 9(28.1%), 14(42.4%) and 18(60.0%), respectively. From the above resutls, it was concluded that the optimal conditions of pH osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOsm and 120 min., respectively,a nd somewhat high conception rate might be resulted from transfer of frozen embryos of morula stage and fresh embryos of blastocyst stage.

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