• Journal of Embryo Transfer
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• v.21 no.4
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• pp.281-286
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• 2006

This study was conducted to investigate the hormonal changes in cultured medium during in vitro culture of bovine oviduct epithelial cells (BOEC) supplemented with interleukin (IL)-4 of 0.001, 0.01, 0.1 or 1 ng/ml. BOEC were collected from the oviduct and washed 3 times with 1% antibiotic-mycotic-DMEM medium and cultured at $39^{\circ}C$, 5% $CO_2$, 95% air for 24$\sim$120 hrs. The cultured media were analyzed hormonal changes with hormonal analyzing kit (progesterone (P4), estradiol (E2) : Perkin Elmer, USA) and Transforming growth factor (TGF)-$\beta$ with Eliza kit (Promega, USA). The production of P4 in 0.001 IL-4 was increased as the culture time increased. P4 production was significantly higher in the medium cultured for 120 hrs than 24 hrs (P<0.05). P4 production in 0.01 ng/ml group was similar to that of 0.001 ng/ml. The production of E2 in 0.001 and 0.01 ng/ml groups were increased to 72 hrs like P4 production and showed significantly different between the culture periods (P<0.05). After the culture for 96 hrs, P4 and E2 production were increased to 96 hrs, but decreased at 120 hrs. The production of TGF-$\beta$ showed no changes according to culture period or supplementation of IL-4. In conclusion, the supplementation of IL-4 can increase the production of P4 and E2 and might have important role for the successful pregnancy in bovine.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.79-79
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• 2002

본 연구에서는 in vitro에서 성숙된 난자의 핵성숙(Polar Body extrusion)에 소요되는 시간과 배반포 단계로의 발달능력 사이의 관계를 비교하여 조기에 발달능력을 가진 embryo를 선발할 수 있는 IVP 체계를 개발하고자 하였으며 in vitro maturation(IVM)에 따른 first polar body(PB) 형성, IVM과 IVF 시간이 oocyte의 발달에 미치는 영향과 생산된 배반포의 세포수를 평가하였다. IVM은 TCM199 배양액을 사용하였고 in vitro fertilization(IVF)은 Fer -TALP용액을 사용하였으며 in vitro culture(IVC)는 CRlaa 배양액을 사용하여 2일까지는 0.3% BSA를 3일 부터는 10%FBS와 bovine oviduct epithelial cell을 첨가하여 배양하였다. IVM 시간에 따른 PB의 출현율은 0hr(0%), 6hr(0%), 12hr(0%), 14hr(8.7%), 16hr(40.5%), 18hr(48.0%), 20hr(65%), 22(68%) 그리고 24hr(74.5%)을 보였으며 IVM 시간에 따른 cleavage 및 8cell 발달율 사이에는 유의적인 차이가 없었으나 배반포(BL) 및 8cell에서 배반포로 발달률은 18시간(BL 31$\pm$6, BL/8cell 82 $\pm$5%)에서 가장 높게 나타났으며 24시간(BL 17$\pm$2, BL/8cell 60$\pm$8%)과 유의적인 차이를 보였다(P<0.05). IVC 7일째 배반포의 총세포수와 trophoblast(TE) 세포수는 IVM 18시간(mean$\pm$S.E.; total: 131.1$\pm$34.0, TE: 97.6$\pm$29.6)에서 24시간(total: 112.2$\pm$17.5, TE: 80.1$\pm$15.6)보다 유의하게 많은 것으로 나왔으나(P<0.05) 7일째의 inner cell mass(ICM) 숫자(18hr 33.5$\pm$12.8 vs 24hr 32.1$\pm$12.0)와 8일째 ICM, TE 그리고 총 세포수에는 유의성 있는 차이가 없었다. IVM 18시간에서 PB 형성과 8cell 발달률 사이에 높은 상관성을 보였고 배반포 및 8cell에서 배반포 단계로 높은 발달률을 보였으며 생산된 배반포의 TE 숫자와 총 세포수가 유의하게 많은 것으로 나타났다. 따라서 IVM 18시간 실시하였을 경우 보다 많은 세포수를 가진 배반포 발달 가능성이 높은 embryo를 조기에 선발 가능할 것으로 사료된다.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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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.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.648-656
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• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.639-645
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• 1997

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplanted embryos. The oocytes collected from slaughterhouse ovaries were matured 24h in TCM199+10% FBS and exposed to $39^{\circ}C$ or room temperature to allow cytoplasmic maturation and gain activation competence. Donor embryos were treated for 12h with $10{\mu}g/ml$ nocodazole or $0.05{\mu}g/ml$ demicolcine to synchronize the cell cycle stage at 26h after the onset of culture. The blastomeres and recipient oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. In the treatment of oocyte activation and cell cycle regulation of donor nuclei, the room temperature exposure and nocodazole treatment group had significant effect on the developmental rates to morula/blastocyst(21.7% vs 12.1~16.7%), but had no significant effect on the fusion rates between donor blastomeres and recipient oocytes. The developmental rates of bovine nuclear transplanted embryos appeared to be higher significantly in mTALP medium under 5% $O_2$ condition and in TCM199 with bovine oviduct epithelial cell under 20% $O_2$ condition(22.2%) than other groups. In embryo transfer of nuclear transplanted embryos, there were no significant differences in calving rates between the use of excellent and good grade donor embryos.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.219-228
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• 1997

To examine the efficiency of nuclear transplantation the influence of electrical preactivation of recipient cytoplasm on the in vitro developmental potentyl in the nuclear transplant rabbit embryos were evaluated. The embryos of 16-cell stage were collected and synchronized to G1 phase of 32-cell stage. The recipient cytoplasms were obtained by removing the first polar body and chromosome mass by non-disruptive microsurgery procedure. The separated G1 phase blastomeres of 32-cell stage were put into the non-preactivated and/or the preactivated recipient cytoplasm by electrical stimulation. After culture until 20h post-hCG injection, the nuclear transplant oocytes were electrofused. The fused nuclear transplant embryos were co-cultured with rabbit oviduct epithelial cells and monitored every 24h to assess for developmental rate. After in vitro culture for 120h, the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 and their blastomere were counted. The electrofusion rate was similar to the non-preactivated and preactivated recipient cytoplasm(81.8 and 85.7%, respectively). However, the in vitro developmental rate to blastocyst stage with the non-preactivated recipient cytoplasm (57.1%) was found significantly (P<0.05) higher, compared to the preactivated recipient cytoplasm(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased significantly (P<0.05) more in the non-preactivated recipient cytoplasm (163.7 cells), as compared with the preactivated recipient cytoplasm(85.4 cells). These results considered better that non-preactivated oocytes, MII phase oocytes, were used for recipient cytoplasms in the rabbit nuclear transplant procedure.

• Korean Journal of Animal Reproduction
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• v.18 no.2
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• pp.121-126
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• 1994

This study was conducted to examine the condition of activation of the nuclear transplant bovine embryos. In vitro fertilized(IVF) and nuclear transplant embryos(NTs) were co-cultured with bovine oviduct epithelial tissue(BOET). NTs were treated with cycloheximide(CHXM) for 0 to 6 h after electrofusion to investigate the activation conditin of recipient ooplast. Then, the infljence of the CHXM treatment timing on the cleavage and development of NTs were investigated in relation to the nuclear transplant time. The cleavage rates of NTs were increased with the increasing time of the CHXM treatment from 0 to 6 h (54.7 to 91.3%, P<0.01). Similar trend was shown in the development into the morula or blastocyst stage, but very limitted. Activation of enucleated oocytes prior to fusion enhanced development of NTs compared with that post fustion. This result suggests that the frequency of activation of NTs can be greatly enhanced by treating with CHXM for 6 h. The result also suggests that if blastomeres of unknown cell cycle stage are used, activation of enucleated oocytes prior to fusion enhances development of NTs.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.161-169
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• 1997

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.647-659
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• 1997

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.13-20
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 $\mu\textrm{g}$/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at $39^{\circ}C, 5% CO_2$ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).