• 한국가축번식학회지
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• 제14권1호
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• pp.43-49
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• 1990

To improve the freezing techniques of animal embryos using vitrification solution as a cryoprotectant rabbit embryos, by cell stages, dehydration temperature and dehydration temperature and dehydratin time, were frozen-thawed and cultured. Following are the main results obtained. 1. The damage rate of zona pellucida after thawing was higher(13.6%) when the cell stage of embryos was less than 4 cells than when the cell stage was 8~16 cell or morula. The damage rate was higher when the dehydration temperature was 4$^{\circ}C$ than -3$0^{\circ}C$ or -50~-8$0^{\circ}C$. The zona pellucida was damaged more when dehydrated for 5 min than when dehydrated for 10~15 min. 2. After being cultured for 72 hours, 5.3% of 4 cell(or less) embryos were developed to morula, while 86.4% of morula embryos were developed further. 3. More percentage of embryos(73.2%) was developed when dehydrated at -3$0^{\circ}C$ than when dehydrated at 4$^{\circ}C$ at -5$0^{\circ}C$~-8$0^{\circ}C$. 4. The hatching rate was higher when dehydrated for 5 min. When the embryos were dehydrated for 10~15 min and cultured for 24 hours, they were not even developed or development was not good in later stages.

• Asian-Australasian Journal of Animal Sciences
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• 제33권10호
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• pp.1579-1589
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• 2020

Objective: This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction. Methods: Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3). Results: LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos. Conclusion: The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.

• Clinical and Experimental Reproductive Medicine
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• 제21권1호
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• pp.77-81
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• 1994

To improve in vitro embryonic cell development, this study was desigend to culture in vitro fertilized early embryos of mouse in two different systems; conditioned medium alone and ampullary cells co-culture. Thirty two of 83 embryos(38.6%) were blocked in the 2 cell stage by co-culture, as compared to forty of 42 embryos(95.2%) in control group for 24hours culture. And all the embryonic cells cultured for conditioned medium alone were blocked for 48 hours culture. Twenty seven of 46 embryos (58.7 %) which overcome culture block in 2 cell stage by cocultured were developed morular and expanded blastocyst, and ninteen of 46 embryos(26.1 %) underwent hatching for 96 hours culture. The cellular fragmented rates for embryo were 26.2% in medium alone; 10 fragmented blastomere were graded mild status and 1 fragmented blastomere in severe status. On the other hand, the fragmented rate for 48 hours co-cultured were 15.7%03/83); 8 fragmented embryos were graded mild status, moderate status in 3 fragmented embryos and severe in 2 fragmented embryos respectively. In conclusion, the co-culture of embryos with ampullary cells is good to improve quality of embryos and overcome of culture block as well as development of cell cleavage.

• 한국가축번식학회지
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• 제20권3호
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• pp.299-305
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• 1996

This study was conducted to investigate the effects of co-culture for the development rate to morula/blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine oviductal epithelial cell monolayers(POEC) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8∼16-cell and morula/blastocyst stage were 57.2, 48.2, 37.2 and 19.3% in Ham's F-10 with POEC, and 51.4, 41.2, 31.1, and 15.5% in TCM-HEPES with POEC, respectively. The above development rates to morula/blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TCM-HEPES with out POEC(P<0.05). The in vitro development rates to the morula/blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without POEC were 1.1∼1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with POEC in the two different media enhanced the development of fertilized eggs to morula/blastocyst stages in vitro. However, we didn't find out any difference for the in vitro development to morula/blastocyst stages between Ham's F-10 and TCM-HEPES media.

• Reproductive and Developmental Biology
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• 제28권1호
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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%).

• 한국수정란이식학회지
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• 제11권3호
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• pp.217-223
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• 1996

This study was conducted to investigate the effects of co-culture for the development rate to morula /blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine endometrial cell monolayers(PEM) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8~16-cell and morula /blastocyst stage were 49.6, 40.5, 28.2 and 15.3% in Ham's F-10 with PEM, and 55.3, 45.9, 32.7, and 17.6% in TCM-HEPES with PEM, respectively. The above development rates to morula /blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TGM-HEPES without PEM(P<0.05). The in vitro development rates to the morula /blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without PEM were 0~1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with PEM in the two different media enhanced the development of fertilized eggs to morula /blastocyst stages in vitro. However, we didn't find out any differences for the in vitro development to morula /blastocyst stages between Ham's F-10 and TcM-HEPES media.

• 한국임상수의학회지
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• 제10권2호
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• pp.243-249
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• 1993

Although plenty of experiments for production of genetically identical twins have been reported, most of these reports were focused on microsurgical bisection of morula stage embryos, and little research has been performed for the production with frozen emb교os. The objective of these experiments were to assess the in vitro and in vitro developmental potential of blastomeres isolated from frozen-thawed 4-cell and 8-cell mouse embryos and to produce the identical multiplets from those embryos. The percentages of isolated 1/4, 2/4, 4/4, (control), 1/8, 2/8, 3/8, 4/8 and 8/8(control) blastomeres of 4-cell and 8-cell mouse emb교os which developed to normal blastocyst were 38.7%, 73.6%, 96.2(control), 15.1%, 40.1%, 65.9%, 88.3%, and 98.5%(control), respectively. The percentages of isolated 1/4, 2/4, 4/4(control), 1/8, 2/8, 3/8, 4/8 and 8/8(control) blastomeres of frozen-thawed 4-cell and 8-cell mouse embryos which developed to normal blastocyst were 35.6%, 68.5%, 100.0%(control), 16.1%, 50.6%, 71.7%, 90.9% and 100.0%(control), respectively. Normal 26 pups were obtained by transfer of 240 blastocyts developed 1, on 1/4 to 8/8 blastomeres. Those 26 pups obtained, 4 identical twins were produced from 2/4, 2/8, 3/8 and 4/8 blastomeres.

• 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.

• 한국가축번식학회지
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• 제17권4호
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• pp.305-310
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• 1994

These expriments were carried out to investigate existence of H-Y antibody in the rat serum immunized against H-Y antigen from rat spleen cells and effect of H-Y antiserum on development of mouse male embryos. The results obtained were summerized as follows : 1. When mouse embryos were cultured for 48∼72 hrs in the Ham's F10 containing 16% of FBS(fetal bovine serum) or RNS(rat normal serum), percentages of embryos developed from 2, 4, 8 and 16-cell embryo to morulae were 20, 27, 94 and 100%, respectively, in FBS and 8, 7, 94 and 100%, respectively, in RNS. Eight to 16-cell embryos showed no difference in development rate between FBS adn RNS. 2. When 8∼16-cell mouse embryos were cultured for 24∼48 hrs in the Ham's F10 containing FBS, RNS＋GPC(guinea pig complement) and RAS(rat antiserum)＋GPC, proportions of embryos developed to the expanded blastocyst stage were 100, 82.4 and 52.1∼53.6%(ave.52.9), respectively, so that it was suggested that rat antiserum suppressed development of male embryos. 3. When 8∼16-cell mouse embryos were cultured for 24∼48 hrs in the Ham's F10 containing FBS, RNS, RNS＋GPC and RAS＋GPC, proportions of embryos developed to the expanded blastocyst stage were 94.5, 90.9, 82.3 and 47%, respectively, and the embryos developed in the medium containing RAS＋GPC seemed to be female. These results indicated that the antisera prepared through immunized against H-Y antigen from rat spleen cell, possessed H-Y antibody which supressed development of male embryos.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.181-186
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• 2005

본 연구에서는 배양 체계(혈청 첨가 TCM199, TALP, CRlaa 및 혈청 미첨가; IVMD101, IVF100, IVMD101)가 체외수정 또는 미세주입된 수정란의 체외 발달에 미치는 효과를 검토하였다. 또한 미세주입에 사용하는 GFP유전자의 양 및 미세주입 수정란에서 형광발현 양상을 검토하였다. 체외수정된 수정란의 $\geq$ 2세포기, 8세포기 및 배반포 도달율이 미세주입 수정란에 비하여 유의하게 높았다. 혈청 미첨가 배지에서의 8세포기 발달율이 수정란의 종류에 관계없이 유의하게 높았으나(p<0.05; $ 3.3\%\;vs.\;15.5\%$ 및 $21.4\%\;vs.\;39.4\%$, respectively), 배반포 도달율은 유사한 경향이었다($2.7\%\;vs.\;2.3\%$ 및 $23.0\%\;vs.\;23.6\%$, respectively). 한편, 2ng/uL 유전자를 미세주입한 수정란의 $\geq$ 2세포기, 8세포기 및 배반포 도달율이 4 및 8ng/uL의 것에 비하여 높은 경향이었다. 미세주입 수정란의 형광발현율은 1세포기가2및 8세포기에 비하여 유의하게 높았으나(p<0.05), 4세포기 및 배반포 단계와는 차이가 없었다.