• 한국임상수의학회지
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• 제9권1호
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• pp.323-332
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• 1992

The present study was carried out to examine the effect of oviduct epithelium and its conditioned medium on e development of early bovine embryos in vitro. Oocytes obtained from ovarian follicles of slaughtered cows were cultured in TCM199 with 10% fetal calf serum for 22-24hrs and then fertillzed in vitro using frozen-thawed semen treated with BO-caffein, BO-BSA(20mM heparin added). Oviduct epithelium was collected in each stage of the estrus cycle and conditioned medium was the medium in which oviduct epithelium in early luteal stage was cultured. In vitro fertilized bovine embryos of 1～2 cell were co-cultured with oviduct epithelium from different estrus cycles, cultured in conditioned medium, and cultured in rabbit oviduct. The cleavage rates of in vitro fertilized early bovine embryos co-cultured with oviduct epithelial cell from early luteal, luteal and follicular phase of estrus cycle(67.2～70.8%) and cultured in conditioned medium(56.7%) were significantly(p<0.05) higher than that of the control(44.2%) The rate of development to morula or blastocyst stage in oviduct epithelial cell co-culture(15.3～32.5%) from three phase of estrus cycles and conditioned medium(14.5%) were significantly(p<0.05) higher than that of the control(5.2%). The oviduct epithelial cell from early luteal phase gave a significantly( p<0.05) higher rate of development to morula or blastocyst stage than both luteal and follicular phase. The results of in vivo culture in rabbit oviduct of early bovine embryos were 52.1% for the cleavage rate and 26.7% for the rate of development to morula or blastocyst stage.

• 한국수정란이식학회지
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• 제28권1호
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• pp.41-48
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• 2013

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.241-245
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• 2005

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that $20\%$ of round spermatid cultured were matured into elongating spermatid after 24 h, and about $10\%$ of round spermatid cultured showed complete elongation (elongated spermatid) within $24\~48$ h of in vitro culture. No further development was observed within $50\~72$ h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.

• 한국수정란이식학회지
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• 제13권1호
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• pp.43-52
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• 1998

The embryogenesis stimulating activity(ESA) had been shown in co-culture of embryos with bovine oviduct epithelial cell(BOEC) and culture in BOEG-conditioned medium. The present study was undertaken to purify and quantify the embryotropic proteins and to determine the optimum concentration of the embryotropic protein for the proper development of embryos. In BOEC-conditioned medium, five major bands of proteins were detected(66, 53, 40, 32 and 24 kDa) by SDS-PAGE. From these proteins, 288pg of protein that had a 32kDa molecular weight was purified by gel filtration column and perfusion chromatography ion-exchange column. When purified protein was supplemented to the in vitro culture media at various concentrations in protein-free media, 2.5$\mu$g /ml supplement group showed significantly higher rates of embryo development into morula /blastocyst stages than other groups(p<0.05). In conclusion, we purified 32kDa protein from BOEC-conditioned medium and this protein showed optimum embryogenesis stimulating effect at 2.5$\mu$g /ml.

• 한국발생생물학회지:발생과생식
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• 제3권1호
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• pp.59-67
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• 1999

초기배아의 발생과정동안 배아와 모체에서 발현되는 여러 cytokine은 착상을 위한 신호물질로 중요한 역할을 한다. 그 중 interleukin-1$\beta$ (IL-1$\beta$)는 배아와 모체간의 상호 신호전달체로서 성공적인 착상을 위한 필수적인 요소로 작용한다고 알려져 있다. 따라서 본 연구에서는 초기배아의 발생과정에 있어서 IL-1$\beta$ 유전자의 역할을 규명하기 위해 생쥐 초기배아에서의 IL-l$\beta$ 유전자의 발현양상을 역전사중합효소연쇄반응을 통해 조사하였고, IL-l$\beta$ 유전자의 발현에 미치는 수란관과 자궁내막세포의 영향을 밝혀보기 위해 공배양방법을 이용하였다. 그 결과 IL-l$\beta$ in vivo 에서는 4-세포기부터 포배기까지, in vitro에서는 상실배부터 부화중 포배기까지 발현하는 양상을 보였다. 또한 수란관과 자궁내막세포와의 공배양시 대조군과 비교하였을 때 실험군에서 IL-l$\beta$ 유전자의 발현이 촉진되었다. 이러한 결과는 IL-l$\beta$ 의 존재가 착상전 초기배아의 발생에 중요한 역할을 한다는 것을 의미한다. 또한 수란관과 자궁내막세포와의 공배양을 통해 IL-1$\beta$ 유전자의 발현이 수란관과 자궁요소에 의해 조절됨을 확인하였다.

• 한국가축번식학회지
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• 제22권2호
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• pp.111-117
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• 1998

This study was conducted to examine the effect of culture supernatant of bovine oviductal epithelial cell(BOEC) on in vitro development of mouse embryos. To obtain the culture supernatant, ampullary epithelial cell, ithmic epithelial cell and ciliated eptithelial cell of bovine oviduct were cultured in Ham's F-10 su, pp.emented with 10% FCS. The development rates of mouse embryos to blastocyst stage were significantly(P<0.05) higher in BOEC-culture supernatant(72.3∼82.3%) than in Ham's F-10(50.7%). The proportions of embryonic development into hatched blastocysts were significantly(P<0.05) higher in ampullary cell supernatant(43.2%), ithmic cell supernatant(48.4%) and ciliated cell supernatant (27.7%) than in Ham's F-10(14.4%). On the other hand, the effect of ciliated cell supernatant was lower than those of other cell supernatants(P<0.05). And there was no difference between ampullary cell supernatant and ithmic cell supernatnat.

• 한국가축번식학회지
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• 제17권3호
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• pp.243-248
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• 1993

Bovine embryos at 2-to 8-cell produced by in vitro matured/in vitro fertilized(IVM/IVF) were cultured in TCM 199 or Synthetic oviduct fluid(SOF) with 10% fetal bovine serum(FBS) or cocultured with cumulus or bovine oviduct epithelial cell(BOEC) in TCM-199 or SOF medium. In experiment 1, the proportions of embryos developed to morula and blastocysts stages in TCM 199 medium were higher when they were co-cultured with cumulus cell(29%) or BOEC(33%) than that of TCM 199 with 10% FBS(12%, P<0.01). In experiment 2, embryos deived from IVM/IVF were cultured in SOF with 10% FBS or cocultured with cumulus cell or BOEC in same medium. The higher development rates of IVM/IVF embryos developed beyond morula stages were obtained in cumulus cell co-culture group(39%) than those of BOEC group(26%) and SOF with 10% FBS group(17%, P<0.01). The present results indicated that the early development of IVM/IVF embryos can be maintained efficienty in SOF with cumulus cell co-culture.

• 대한수의학회지
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• 제33권1호
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• pp.179-188
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• 1993

The developmental capacity of bovine oocytes under three different culture systems was investigated in this experiment ; One was culture in TCM199 with bovine oviductal epithelial cells(BOEC) for in vitro culture, another was culture in TCM199 with BOEC for 2 days and then transfer of 4~8cell embryos to rabbit oviduct(RO) and the other was transfer of 1 or 2cell embryos to RO for in vivo culture. And the other concern of this experiment was to investigate the effect of culture period and transfer site on recovery. Immature bovine oocytes were cultured in TCM199 with granulosa cells for 22-24hrs and then fertilized in vitro using frozen-thawed semen treated with BO-caffine and BO-BSA. Fifteen to 18hrs after in vitro fertilization oocytes were cultured in TCM199 with BOEC or transferred to RO for 5 days. The rate of development to the morula or blastocyst was higher in transfer of 1 or 2cell embryos to RO(23.1%) than culture in TCM199 with BOEC(11.7%). But, there was no difference between transfer of 1 or 2cell embryos and transfer of 4~8cell embryos to RO(12.8%). Recovery under different culture periods in RO was significantly higher in 90~95hrs(70.1%) than 122~125hrs(50.9%, p<0.05) and recovery significantly increased when oocytes were transferred deeper in RO(2.5cm>, 47.7% ; 2.5~4.5cm, 63.9% ; 4.5cm<, 77.3%, p<0.05). The results show that transfer of 1 or 2cell embryos to RO is an effective means of supporting the further development of in vitro matured and fertilized bovine oocytes than culture in TCM199 with BOEC or transfer of 4~8cell embryos to RO, and recovery from RO increases when oocytes are transferred deeper and incubated shorter in RO.

• 한국수정란이식학회지
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• 제13권3호
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• pp.227-233
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• 1998

난관상피세포와 그 배양액에서 분비된 고분자 분획이 소정자의 수정능력에 어떠한 영향을 미치는지 알아보고자 실시한 실험에서 다음과 같은 결론을 얻었다. 1. 난관상피세포배양액으로부터 MW 5 kDa cut-off bucket을 사용하여 탈염 및 농축을 실시, 단백질/고분자분획을 회수하고 이를 체외수정용 배양액에 첨가하고 난관상피세포 monolayer와 공배양을 통해 체외수정을 실시한 결과 고분자분획첨가 및 난관상피세포 공배양(OM+OEC)군이 고분자분획첨가(OM) 군에 비하여 유의적으로 높은 분할율을 나타내었으며 난관상피 세포 공배 양(OEC)군 및 OM 군 모두 대조군에 비해서는 유의적으로 높은 분할율을 나타내었다(p〈0.01). 2. 난관상피세포와 전배양한 정자의 체외수정능을 조사한 결과 정자와 OEC를 4시간 동안 전배양한 후 체외수정한 군이 난자와 정자를 동시에 배양한 군에 비하여 유의적으로 높은 분할율을 나타내었다(p〈0.01). 이상의 결과에서 소 정자의 체외수정능은 난관 상피세포와의 접촉 및 분비액유래 고분자단백분획의 공동효과에 의한 것으로, 난관상피세포와의전배양은 체외에서 수정능획득을 유도하는 것으로 판단된다.

• 한국가축번식학회지
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• 제17권2호
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• pp.133-139
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• 1993

The studies were carried out to investigate the effects of co-culture with cumulus cell, oviduct epithelial cells and uterine endometrial cells on the in-vitro fertilization and cleavage rate of porcine follicular oocytes. The ovaries were obtained from slaughtered swine. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation. The results obtained in these experiments were summarized as follows : 1. The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with cumulus cells in TCM-199 meidum were 64.6%~74.5% and 37.5%~55.3%, respectively. And in-vitro fertilization rate of cumulus-enclosed oocytes(51.5%) were significantly(p<0.05) higher than cumulus-denuded oocytes(21.7%). 2. The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml oviduct epithelial cells in TCM-199 medium were 53.5% and 37.2%, 61.7% and 46.8%, 54.5% and 31.8%, 42.2% and 26.7%, respectively. 3. The in-vintro maturation and fertilization rate of porcine oocytes co-cultured with 1$\times$106/ml, 1$\times$108/ml, 1$\times$1015/ml uterine endometrial cells in TCM-199 medium were 54.3% and 39.1%, 58.3% and 43.8%, 55.5% and 33.3%, and 45.7% and 30.4%, respectively. 4. When the in-vitro fertilized oocytes were co-cultured with porcine cumulus cells, ovdiduct epithelial cells and uterine endometrial cells, the development rate to the blastocyst stage was 9.5%, 10.7% and 11.8%, respectively and the rates were higher than that of control, 2.1%(p<0.05).