• Development and Reproduction
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• v.3 no.1
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• pp.59-67
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• 1999

To investigate the role of interleukin-l$\beta$ (IL-1$\beta$) in the embryonic development, in vivo and in vitro expression patterns of IL-1$\beta$ gene in the preimplantation mouse embryos were examined by RT-PCR, and the effects of explanted mouse ovi-duct and uterine epithelial cells on the expression of IL-1$\beta$ gene in the pleimplantation mouse embryos were examined by co-culture. IL-1$\beta$ mRNA was detected in the embryos from 4-cell stage to blastocyst stage in vivo and from morula stage to hatching blastocyst stage in vitro. This transcript was not detected from the GV stage to late 2-cell stage in vivo, and not at the 4-cell and 8-cell stages in vitro. For the co-culture of late 2-cell embryos with the explanted mouse oviduct and uterine epithelial cells, oviducts and uterine epithelial cells were isolated at 48 hour alter the hCG injection. The explanted oviduct and uterine epithelial cells in co-culture groups facilitated the IL-1$\beta$ gene expression of the mouse embryos in comparison with the control. Taken together these results suggest that the presence of IL-1$\beta$ plays an important role in preimplantation embryonic development. In addition, the up-regulation of IL-1$\beta$ gene expression by the explanted oviduct and uterine epithelial cells demonstrates that embryonic expression of IL-l$\beta$ gene may be regulated by the interaction with oviductal and uterine factor (s).

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.59-64
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• 2006

This study was conducted to establish a convenient freezing method of boar semen. Boar semen was cooled until $5^{\circ}C$ for 3 hrs using cell freezer and loaded into straws. Semen straws were frozen in different steps in strofoam box filled with $LN_2$. Highest sperm viability (54.0%) was obtained by 1-step freezing(holding at 10 cm height from the surface of $LN_2$ for 10 min). Sperm viability increased by holding at $-102^{\circ}C$ for 10min (74.0%, P<0.05). In thawing regime, sperm viability was significantly higher in $37^{\circ}C$ group than in $52^{\circ}C$ group. The sperm characteristics did not differ between 1-step and 3-step. After IVF using frozen-thawed boar semen, developmental rate of embryos to the morula+blastocyst stage was in 1-step freezing group than that of 3-step freezing group (27.5 vs 14.7%, P<0.05). The result shows that the 1-step freezing with holding at $-102^{\circ}C$ for 10min before plunging into $LN_2$ is a convenient and easy freezing method for boar semen.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.371-379
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• 2001

The objective of this study was to establish an effective cryopreservation method of in vitro-produced bovine embryos. For the vitrification, in virtro-produced embryos at 8-cell, morula and blastocyst stages were exposed to freezing solution containing 5.5 M EG (EG 5.5) for 20 sec, loaded on each containers such as EM grid, OPS and Cryo-loop, and then immediately plunged into liquid nitrogen at -196$^{\circ}C$. Thawed embryos were serially diluted in 0.5, 0.25 and 0.125 M sucrose in m-HPBS, each for 1 min, and cultured in CRlaa medium supplemented with 10% FBS. Significant differences in the rates of re-expanded and hatched embryos were not observed among these embryo containers. The total cell number of expanded blastocyst cultured in vitro after vitrification was examined by Hoechst staining. There were no differences between non-vitrified (180.0 $\pm$ 5.4) and vitrified groups (178.0 $\pm$ 7.5). In addition, when the cellular injuries after vitrification were compared by double staining. There were no significant difference in the ratio of live and dead cells between non-vitrified group (176 : 4) and vitrified group (172 : 6). Therefore, these results suggest that bovine embryos can be cryopreserved easily, effectively and successfully by vitrification using various containers, such as EM grid, OPS or Cryo-loop in the presence of EG 5.5 freezing solution.

• Korean Journal of Ichthyology
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• v.27 no.4
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• pp.300-305
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• 2015

The characteristics of 11 spawning sites in the natural environment of the Korean endemic species, Liobagrus somjinensis were investigated in the Yocheon (stream) of Seomjingang (river), located in Sikjeong-dong, Namwon-si, Jeollabuk-do, Korea from May to June 2015. The spawning sites had largely two parts, the upper region close to the surface of the waters and under region at bottom: the upper part was covered with a large and flat boulder over about 256 mm in size, whereas the under part consisted mainly of pebbles and sand. The large boulder put on the upper region may be seen as a bulwark for guarding eggs laid at the under (bottom) region. All the sites under the boulder showed a similar structure having a small hollow filled with an egg mass and an individual (male). The spawning bottoms left clean having no any benthic invertebrates and detritus. The egg shape was spherical, yellow and covered with a transparent membrane and the egg size was $3.31{\pm}0.15mm$ (n=30) in diameter. The development stage of each egg mass under the boulder was almost same from morula stage to formation stage of eye lens. The average number of eggs in 11 spawning sites was $121{\pm}35.5$ (84~176) and the average number of female fecundity in ovary was $143{\pm}31.3$ (110~232).

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.1-7
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• 2000

Objective: Mammalian embryos undergo changes of energy environment for transfer from oviduct to uterus. Also, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration (oviduct - glucose: 0.5 mM, pyruvate: 0.32 mM, lactate: 10.5 mM; uterus - goucose: 3.15 mM, pyruvate: 0.1mM, lactate: 5.87 mM, respectively). This study was conducted to examine the effect of these energy sources added in DMEM with glutamine on the mouse embryo development. Methods: There was used ICR female mouse. Two cell embryos of mouse are collected by method of 'flushing'. Flushing fluid was used Ham's F-10 added to 20% FBS. The collected 2 cell embryos were cultured in media such as Control (only DMEM), group A and B (DMEM supplemented with 0.5 mM and 3.15 mM glucose), and group C and D (DMEM supplemented with 0.1 mM and 0.32 mM pyruvate), and group E and F (DMEM supplemented with 5.87 mM and 10.5 mM lactate). All experimental media supplemented with 20% hFF, respectively. Pattern of embryo development was observed to interval at 24hr during 96hr. Results : The media with glutamine added glucose (group A: 51.0%; group B: 48.4%) was significantly (p<0.05) higher than other experimental group in development into the morula stage after 24 hr in culture, but not significantly different compared with control and the rate of development into the blastocyst was significantly (p<0.05) low in the both of pyruvate (group C: 7.9% group D: 6.8%) and lactate (group E: 7.1%, group F: 7.1%) treatment group after 48 hr in culture. Development into the blastocyst and hatched balstocyst after 72 hr in culture revealed similarly in control (81.9%) and glucose treatment group (group A: 83.3%, group B: 82.8%). However, development into the hatched and attached blastocyst after 96hr in culture revealed significantly (p<0.05) development in the glucose treatment group (group A: 82.3%, group B: 78.5%) than control (63.2%), and its of pyruvate (group C: 34.1%, group D: 34.1%) and lactate (group E: 25.9%, group F: 33.3%) treatment group were significantly (p<0.05) lower than control similar to previous observations. Conclusion : The glucose added to the DMEM with only glutamine, as energy source, was highly to the rate of development compared with control, but the other energy sources were not, synthetically. Above refer to, the human reproductive organ (oviduct, uterus) contains energy sources of different concentration. Thus, further studies are will examine continuously to effects by interaction of different energy sources in the mouse embryo development, and these results will provide to foundation on the human embryo culture.

• The Korean Journal of Zoology
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• v.39 no.4
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• pp.352-361
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• 1996

The c-myc proto-onco9ene, one of the immediately early genes, is involved in ceflular proliferation and differentiation, and its biologleal function is regulated hy dimerization with a heterodimeric partner, myn. In the present study, gene expression patterns of c-myc and myn during mouse preimplantation embryo development were examined using a semi-quantitative reverse transcription-poiymerase chain reaction (RT-PCR). Myn transcripts were rather constitutively expressed throughout embryonic stages with a slight increase only at biastocyst stage. in contrast, expression of c-myc transcripts wm developmental stage-'pedfic. The c-myc transcripts were detected at 1-cell stage, declined abruptly at 2-cell stage and then increased gradually at blastocyst stage. To examine the possible role of myn during preimpiantation mouse embryo development, two myn antisense oligonucleotides spanning the tail of zipper dognain (myn2; 20-mer) and the second helix domain (myn3; 20-mer) were microinjected into the fertilized 1-cellembryos. Microinjection of myn2 and myn3 resulted in developmental tion at morula/biastocyst transition stage, leading to the fiagentation of embryos. Talien together, these results suggest that c-myc and its heterodimeric partner, myn, are differentially expressed In a developmental stage-dependent manner, and myn may play an important role in mouse preimpiantation embryo development.

• Journal of Life Science
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• v.9 no.3
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• pp.333-340
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• 1999

The toxic effect of TBTO on Chlorella and Rotifer was observed. The value of 48hr-LC50 for Chlorella (3.3$\mu\textrm{g}$/L) estimated to be almost 500 times as high as that for Rotifer (6.7ng/L). A fertilized egg of olive flounder exposed in an embryo-formation stage was mostly influenced by TBTO toxicity when the fertilized egg at each stage until hatching was exposed to TBTO at the concentrations of 5 to 200ng/L. The values of LT50 were estimated to be 68.0, 41.0, 21.0, 13.0, 7.7 and 4.7 hours at 5, 10, 25, 50, 100 and 200ng/L of TBTO, respectively when the fertilized egg in a morula stage was exposed to TBTO, and the 48hr-LC50 was 8 ng/L. In case of TBTO treatment in an embryo-formation stage, the values of LT50 were 33.0, 12.5, 3.5, 1.3, 0.5 and 0.2 hours at 5, 10, 25, 50, 100 and 200ng/L of TBTO, respectively, and the value of 48hr-LC50 was 4ng/L. The values of LT50 were estimated to be 17.0, 11.0, 6.2, 4.0, 2.6 and 1.7 hours at 5, 10, 25, 50, 100 and 200ng/L of TBTO, respectively when the fertilized egg in a stage just before hatching was exposed to TBTO, and the 48hr-LC50 was below 1ng/L. The percentages of hatching were 46.2, 20.6, 21.9, 20.6 and 13.2% at 25, 50, 100, 250 and 500ng/L of TBTO, respectively when the fertilized egg in the stage just before hatching was exposed to TBTO and the measurement was done at second day after the completion of hatching. However no survival after the completion of hatching was found in all cases. With the treatment of 1, 5 and 10ng/L of TBTO, the percentages of hatching were 80.5, 70.0 and 44.1%, respectively. The percentages of survival until second day after the completion of hatching were 80.0, 63.3 and 9.1%, respectively. The percentages of hatching and survivability after the completion of hatching for the control were 84.5 and 82.5%, respectively.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.49-57
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• 1991

This study was carried out to investigate the effect of equilibration time, precooling and straw loading method on the post-thaw survival rates of mouse embryos cryopreserved by vitrification method. The effect of the vitrification procedure on the damage of the embryos was also examined by the straining of nuclei using Hoechst 33342. The results obtained were summarized as follows; 1. The equilibration in Medium-1 for 10 minutes was considered to be the optimum equilibration time. Embryos equilibrated in Medium-1 for 10 minutes(81.0%) showed significantly(P<0.05) higher survival rates than those equilibrated for 5 minutes(40.0%) or 15 minutes(74.1%). 2. The survival rate of embryos cryopreserved using the double Medium-2 column(81.0%) was significantly(P<0.01) higher than that using the single Medium-2 column, whish was considered to be due to the double Medium-2 column method being more reliable for preventing contamination of diluent solution of 1M sucrose. 3. The survival rate of compacted morula stage embryos cryopreserved with the precooled and non-precooled Medium-2 was not significantly(P<0.05) different. 4. The number of blastomeres of embryos at late blastocyst stage after culture of mouse morulae for 24 to 28hours was significantly(P<0.05) decreased by freezing embryos using vitrification(53.3${\pm}$1.6 vs 41.4${\pm}$1.5), which was considered to be due to the damage of embryos during vitrification and the delay of embryo development by handling in vitro. 5. The vitrification procedure is considered to be a very simple and efficient method for cryopreservation of embryos at early developmental stage.

• Korean Journal of Animal Reproduction
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• v.16 no.2
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• pp.117-123
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• 1992

This Study was carried out ot investigate the effects of concentration and equilibration time of cryoprotective aagents on survival rate of slowly and ultrarapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotective agents and 0.25M sucrose were slowly freezed(from 2$0^{\circ}C$ to -7$^{\circ}C$/-1$^{\circ}C$/min., from -7$^{\circ}C$ to -35$^{\circ}C$/-0.2$^{\circ}C$/min., from -35$^{\circ}C$ to -38$^{\circ}C$/-0.3$^{\circ}C$/min.) by Cell Freezer and directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water bath. Survival rate was defined as development rate to the morula and blastocyst stage after in vitro culture or by FDA test. The results are summarized as follows : 1. The survival rates of porcine embryos after slow frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 80.6, 84.7, 75.0 or 78.8%, respectively. 2. The survival rates of porcine embryos after slow frozen-thawing in the freezing medium of 0.50M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 80.9, 82.4, 73.1 or 77.1%, respectively. 3. The survival rates of porcine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0M glycerol, 3.0M DMSO, 2.0M propanediol or 2.0M glycerol＋2.0M propanediol was 65.3, 68.6, 63.2 or 59.9%, respectively. 4. The survival rates of porcine embryos after ultrapid frozen-thawing in the freezing medium of 0.50M sucrose added 2.0M glycerol, 3.0M DMSO, 2.0 propanediol or 2.0M glycerol＋2.0M propanediol was 67.5, 62.9, 56.9, or 62.8%, respectively. 5. The higher survival rate of porcine embryos was attained at the short period ofequilibration time(5min.) in the freezing medium added 0.25M sucrose and 3.0 DMSO compared to those of 10 or 20min. equilibration time in the same condition.

• Korean Journal of Animal Reproduction
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• v.16 no.2
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• pp.125-131
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• 1992

This study was carried out to investigate the effects of concentration and equilibration time of cryoprotective agents on the survival rate of slowly and ultrarapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water bath. Survival rate was defined as development rat to the morula and blastocyst stage after in vitro culture or by FDA test. The results are summarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0, 2.5, 3.0, 3.5, or 4.0M glycerol was 65.3, 61.8, 64.3, 59.4 or 39.4%, respectively. Addition of 0.25M sucrose into the freezing medium containing 2.0M glycerol showed higher survival rate than those of 2.5~4.0M glycerol. 2. The survival rates of porcine embryos after ultraradpid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0, 2.5, 3.0, 3.5 or 4.0M DMSO was 65.6, 67.6, 68.6, 60.6 or 23.6%, respectively. However, addition of 0.25M sucrose into the freezing medium containing 3.0M DMSO showed higher survival rate than those of 2.0, 2.5, 3.5 or 4.0M DMSO. 3. The survival rates of porcine embryos after ultrapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0, 2.5, 3.0, 3.5 or 4.0M propanediol was 63.2, 60.3, 62.1, 52.3 or 24.3%, respectively. Addition of 0.25M sucroese into the freezing medium containing 2.0M propanediol showed higher survival rate than those of 2.5~4.0M glycerol. 4. The survival rates of porcine embryos after ultrarapid frozen-thawing the freezing medium of 2.0M glycerol added 0.10, 0.25, 0.50 or 0.75M sucrose was 61.8, 70.8, 67.6 or 52.2%, respectively. Addition of 2.0M glycerol into the freezing medium containing 0.25M sucreose showed higher survival rate than that those of 0.10, 0.50 or 0.75M sucrose. 5. The higher suvival rate of porcine embryos were attained at short period of equilibration time 92.5~5min.) in the freezing medium added 0.25M sucreose and 3.0M compared to those of 10 or 20min. equilibration time in the same condition.