• 한국가축번식학회지
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• 제18권2호
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• pp.105-111
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• 1994

The effects of human or mouse leukemia inhibitory factor(hLIF or mLIF) were examined as a means of increasing the development of in vitro matured(IVM) and in vitro fertilized (IVF) oocytes into morulae or blastocysts. Cell numbers of blastocysts were also counted using Hochest dye staining. Two-to 8-cell embryos derived from bovine IVM/IVF oocytes were cultured 5 to 6 days in CRI aa with or without mLIF or hLIF. All culture media were contained 3mg/ml bovine serum albumin. In experiment 1, the proportion of embryos developed to morulae and blastocysts in CRI aa containing 5,000U/ml mLIF(37.8%) was slightly higher than those of CRIaa containing 1,000U/ml mLIF(34.6%) and 0 U/ml mLIF(27.4%; P>0.05). In experiment 2, 0, 1,000 and 5,000U/ml of hLIF added to CR1aa media yielded 27.6%, 43.0% and 35.5% morulae and blastocysts, respectively(p>0.05). These were no significant increases in cell number among treatments(p>0.05). These results were indicating that mLIF or hLF can increase the proportion of embryos that develop into morulae and blastocysts without and increase in the cell number.

• 한국수정란이식학회지
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• 제11권2호
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• pp.151-158
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• 1996

This study was carried out to investigate the effect of equilibration time, sucrose concentration and age of embryo on survival and developmental rates of bovine IVF expanding blastocysts frozen-thawed by direct transfer method. The bovine oocytes were collected from 2~5mm follicles, matured for 20~24hrs in 5% $CO_2$incubator and then fertilized with frozen-thawed semen. Expanding blastocysts at day 7, 8, 9, 10 and 11 after IVF were frozen in 1.8M ethylene glycol(EG). Survival and hatching rates of frozen-thawed IVF embryos were examined. The results were as follow ; Survival and hatching rate of TVF expanding blastocysts after 10, 20, 3Omin exposure in 1.8M EG were 100,0,90.9, 47.1, 85.0, 75.0 and 62.5% respectively. Survival rates of IVF expanding blastocysts frozen with 1.8M EG and various concentration(0, 0.25, 0.5, 1M) of sucrose were 73.3, 25. 0, 16.7, 9.1% respectively. Survival and hatching rates of IVF expanding blastocysts frozen-thawed according to age of embryo(Day 7, 8, 9,10, 11) were 86.1, 84.8, 79.3, 61.4, 51.3, 74.2, 76.9, 71.7, 63.0 and 65.0% respectively. In conclusion, the age of the embryo(Day 7, 8) is very important for the successful freezing of IVF bovine embryos and 1.8M ethylene glycol not containing sucrose may be effective cryoprotectant for direct transfer method.

• 한국가축번식학회지
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• 제20권3호
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• pp.279-288
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• 1996

본 연구는 EGF가 체외성숙과 수정에 의해 생산된 소 수정란의 발달과 inner cell mass (ICM)와 trophectoderm (TE) 세포수에 미치는 영향 및 공동배양시의 첨가효가를 조사하고 그와 더불어 간적면역 형광법을 이용하여 EGF-R 단백의 발현 유무를 조사하기 위해 실시하였다. 그 결과를 요약하면 다음과 같다. EGF 1, 10, 100 ng/ml의 농도로 처리되었던 4-세포기와 8-세포기 배는 대조군에 비하여 유의차는 인정되지 않았으나 양호한 배반포기 배 발달과 ICM과 TE 세포수 증가 양상을 나타내었다. 특히, 발달단계에 따른 EGF (10ng/ml) 효과를 조사하였던 바, 8-세포기 이후 배에서 대조군에 비하여 배반포기까지 유의한 배 발달을 유도하는 것을 확인할 수 있었지만 (p<0.05), ICM과 TE 세포수 증가에는 유의한 영향을 미치지 못하는 것을 알 수 있었다. 또한, 간접 면역 형광에 의한 EGF-R의 발현 유무를 조사한 결과, EGF-R는 4-세포기 이후에 발현되며 그 강도는 발달단계가 진행되면서 다양하게 나타난다는 것을 알 수 있었다. 한편, 수정란과 난구세포 공동배양 군은 EGF의 첨가 유무에 상관없이 대조군에 비하여 유의한 배 발달과 총세포수의 증가를 나타내며, 공동배양군에 대한 EGF 첨가는 수정란과 난구세포와의 공동배양 효과를 증진시키는 것으로 나타났다. 따라서, EGF는 착상전 소 수정란의 4-세포기 이후에 발현디는 EGF-R에 반응하여 배 발달을 유기하고, 공동배양시의 배 발달에 유용한 물질형성을 촉진시키지만, 배반포기 배의 ICM과 TE 세포수 증가에는 유의한 영향을 나타내지 못한다는 것을 알 수 있었다.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.78-78
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• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• 한국수정란이식학회지
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• 제12권3호
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• pp.277-282
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• 1997

The purpose of this experiment was to determine the effects of thiol compounds, $\beta$-mercaptoethanol($\beta$-ME) and cystearrone with buffalo rat liver cell(BRLC) co-culture on the development and intracellular glutathione(GSH) concentrations of bovine embryos produced by in vitro inaturation(IVM) and in vitro fertilization(IVF). Bovine IVM /IVF embryos developed to 2~8 cell stage were co-cultured with BRLC in GRlaa with or without thiol compounds. The developmental rate beyond morulae stage in CRlaa containing 0, 10,25 and 50$\pi$M $\beta$-ME with BRLG were 63.0, 74.0, 72.3 and 77.1%, respectively. And the developmental rate with 0, 25, 50 and 75$\pi$M cystearnine with BRLC were 69.6, 77.6, 81.0 and 76.8%, respectively. The developmental rate beyond morulae stage of GRlaa containing thiol compound with BRLG group was higher than that of control group. The intracellular GSH concentrations of blastocysts cultured for 5 days in GRlaa containing 0 and 50$\pi$M $\beta$-ME or cysteamine with BRLG were 81.2 and 86.4, 83.2 and 84.2pM, respectively. The intracellular GSH concentrations of blastocysts in GRlaa containing thiol compounds with BRLG was slightly higher than that of control group The cell numbers of blastocysts were not difference in all experimental groups. These results indicate that thiol compounds with BRLG co-culture was increased the percentage of developed into morulae and blastocysts, and intracellular GSII concentrations of blastocysts embryos.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.221-226
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• 2004

The present study was performed to investigate the effects of in vitro maturation (IVM) and in vitro fertilization (IVF) duration on the development of Korean Native Cattle embryos. The time of blastocyst formation and the quality of blastocysts based on cell numbers were examined. The cleavage rate increased with the length of IVF duration in the groups of 18-hr IVM, but was constant in the groups of 24-hr IVM. The development rate to the 8-cell stage was significantly higher in the IVM 18: IVF 20 group than in the IVM 24: IVF 24 group. The development rate to the blastocyst stage was highest in the IVM 18: IVF 20 group, significantly different from that of the IVM 18: IVF 16, IVM 24: IVF 20 and IVM 24: IVF 24 group. The time of blastocysts formation tended to be shorter when IVM and IVF duration were decreased. The number of inner cell mass, trophoblast and the total cells were significantly higher in the IVM 18: IVF 16 group than in the IVM 24: IVF 24 group (P<0.05). These results demonstrated that the IVM and IVF duration should be adequate for the efficient production of bovine embryos, and it might particularly be essential to determine the proper combination of IVM and IVF duration.

• 한국가축번식학회지
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• 제21권4호
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• pp.335-343
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• 1997

The effect of thiol compounds on development and intracellular glutathione(GSH) concentrations of bovine embryos produced by in vitro maturation and in vitro fertilization(IVM/IVF) was examined in CRlaa medium with or without $\beta$-mercaptoethanol(0, 10, 25 and 50$\mu$MME) and cysteamine(0, 25, 50 and 75 $\mu$M). Numbers of cells comprising blastocysts were also counted using double fluorescence stain and the total glutathione levels(oxidized and reduced form) of morula and blastocyst embryos were than measured by an enzymatic method. Following routine IVM/IVF procedures oocytes and zygotes were cultured for 40 to 44h in CRlaa medium. Then 2 to 8-cell embyos had cumulus cell removed and were allotted randomly to the experimental medium. In Experiment 1, the proportion of embryos developing to and beyond morulae stages in 0, 10, 25 and 50 $\mu$M $\beta$-ME was 42.9%, 50.0%, 53.7% and 65.6%, respectively. Fifty $\mu$M $\beta$-ME group was significantly higher than those of any other groups (P<0.05). In Experiment 2, the percentages of embryos developed beyond morulae stages in 0, 25, 50 and 75 $\mu$M cysteamine was 42.9%, 40.4%, 60.0% and 59.2%, respectively. Fifty and 75$\mu$M cysteamine groups were significantly higher than in 0 and 25 $\mu$M cysteamine groups, but all of culture medium containing cysteamine(52.6%) was not significantly difference in control group(42.9%). In Experiment 3, the intracellular GSH concentrations of morulae and blastocyst embryos in 0 and 50 $\mu$M $\beta$-ME was 42.4 pM and 44.9 pM, 49.5 pM and 67.8 pM, respectively. Morulae embryos were not difference, but blastocyst embryos were significantly difference between treatments(P<0.05). In Experiment 4, the intracellular GSH concentrations of morulae in CRlaa with or without cysteamine were 39.8 pM and 45.6 pM, and blastocysts were 59.3 pM and 66.8 pM, respectively. Cell numbers of blastocysts were similar to in all experimental groups. These experiments indicate that thiol compounds can increase the proportion of embryos that developing to and beyond morulae stage and the intracellular GSH concentrations.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.48-48
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• 2002

Chimerism has become an important tool for investigating fundamental aspects of early embryonic development and differentiation in mammals for producing transgenic animals. The objective of this study was to evaluate the developmental capacity of chimeric embryos reconstructed with parthenotes and IVF bovine embryos into empty zona pellucida. The MII oocytes were activated by two treatment groups [Group 1, 5 μM inomycin, 5min, ＋ 10 ㎍/㎖ cycloheximide (CHX)/5 ㎍/㎖ cytochalasin B (CCB), 3 h; Group 2, 5 μM ionomycin, 5 min ＋ 1.9 mM 6-dimetylaminopurine (6-DMAP), 3 h]. (omitted)

• 한국가축번식학회지
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• 제26권4호
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• pp.329-338
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• 2002

This study was carried out to investigate the developmental rates of embryo reconstructed with different cell type and to estimate correlation of transcriptional level of octamer-binding transcription factor 4 (Oct4) and fibroblast growth factor 4 (FCF4) gene on peri-implantation stage embryos. Donor cells were transferred into perivitelline space of enucleated oocytes. The karyoplast-cytoplast couplets were accom- plished by cell to cell fusion and activated with ionomycin and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CR 1 aa medium. There is no difference in blastocyst formation rate following nuclear transfer UT) with fetal fibroblast cell (16/50; 32.0%), cumulus cell (16/49; 32.6%) and ear cell (17/52; 32.6%). The expression level of Oct4 and FCF4 in peri-implantation bovine embryo derived from in vitro fertilization (IVF) and NT were determined by reverse-transcription polymerase chain reaction (RT-PCR) technique. In peri-implantation of IVF result in a transient increased of FCF4 paralleled by an increased expression of Oct4. However, Oct4 gene was highly expressed in hatching blastocysts derived from NT compared to IVF. Also, FGF4 expression level in hatching blastocysts and outgrowth stage derived from NT was lower than that of IVF. In conclusion, it is suggested that the different transcription patterns observed in nuclear transfer embryos may lead to a lower rate of embryo development, implantation and pregnancy.

• 한국가축번식학회지
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• 제17권3호
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• pp.193-199
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• 1993

Bovine follicular oocytes were matured in two different conditions, TCM 199＋10% FBS with or without hormones (0.01 unit/ml ovine follicle stimulating hormone, 0.01 unit/ml ovine luteinizing hormone and 1$\mu\textrm{g}$/ml $\beta$-estradiol). There was no significant difference in maturation and fertilization rates of the oocytes between two groups. The result indicates that hormonal treatment does not have beneficial effect on in vitro maturation and fertilization of follicular oocytes. IVF-derived cone-cell bovine embryos were injected with foreign DNA (CChcLf) by microinjection method and then co-cultrued with bovine oviductal epithelial cells. Developmental rate of microinjected embryos to blastocyst stage (21%) was similar to that of non-injected embryos(29%). This result represents that microinjected bovine embryos produced in vitro have a potential of development to normal blastocysts.