• 한국자원식물학회지
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• 제17권2호
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• pp.161-168
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• 2004

Ferritin light heavy chain (FLHC) gene는 일부 중금속과 결합, 저장 및 운반하여 무독화 시킬 수 있는 것으로 알려져 있다. Fe 관련 유전자인 FLHC유전자를 식물 발현용 promoter인 35S promoter와 Tnos를 사용하여 식물 형질전환용 vector를 재조합하였다. 식물세포형질전환용 binary vector는 상기 cassette vector가 조립이 매우 양호하며 border sequence를 가지고 있는 pRD400 binary vector를 사용하여 최종적으로 가나마이신 내성 유전자 (NPT II gene)와 tadpole ferritin heavy chain gene 및 human ferritin light chain gene를 함유하고 있는 binary vector를 재조합하였다. Binary vector의 아그로박테리움에 도입은 triparental mating 방법에 의하여 수행하여 AB배지 및 가나마이신 함유 배지에서 disarmed Ti-vector를 가지고 있는 Agrobacterium tumefaciens MP90/FLHC을 선발하였다. FLHC 유전자 도입된 식물형질전환용 binary vector를 이용하여 형질전환방법을 변형하여 많은 embryo를 유도하였으며 유도된 embryo들은 GA 10mg/L가 첨가된 배지에 지상부를 유도하였다. 형질전환체식물체의 정상적인 생장을 유도하기 위해 최적의 배양조건을 조사하였던 바, 비교적 1/3 MS배지에서 뿌리의 생장과 지상부의 생장이 균일하게 생장하는 경향을 보였으며, 뿌리와 줄기가 잘 발달된 약 7cm의 유식물체를 대량으로 증식하여, 모래와 흙이 1:1로 혼합된 토양에 옮겼다.

• 한국수정란이식학회지
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• 제9권3호
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• pp.249-254
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• 1994

This study was undertaken to investigate effects of granulosa cells on mejotic maturation of porcine oocytes in vitro. The results obtained in this study were summarized as follows : The germinal vesicle breakdown(GVBD) rates were 91.5, 93.3 and 96.6%, respectively, when the cumulus oocy:e cornplexes(COC) in the TCM-199 medium with sodium bicarbonate, Na pyruvate, penicillin G, streptomycin sulfate and 10% FCS were cultured in the condition of FSH(0.02 Au/ml), LH(10 $\mu$g/ml) and FSH + LH added. And when the COC were co-cultured with granulosa cell (5$\times$ 106 cells /ml) in the condition of FSH, LH and FSH + LH added, GVBD rates were 94.3, 92.9 and 98.9%, respectively. However, when the COC were cultured in the condition of hormone free and co-cultured with granulosa cells in the condition of hormone free, the GVBD rates were 40.4 and 86.3%, respectively. The GVBD rates were 41.0, 62.7, 84.6, 88.1 and 93.6%, respectively, when the COC were co-cultured with granulosa cells that the concentrations are 0 cells /ml, 1 $\times$ 106 cells /ml, 5:: 106 cells /ml, 1$\times$ 107 cells /ml and 5$\times$ 107 cells /ml.

• Clinical and Experimental Reproductive Medicine
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• 제42권3호
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• pp.111-117
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• 2015

Objective: In order to increase the number of mature oocytes usable for intracytoplasmic sperm injection (ICSI), we aimed to investigate the effect of co-culturing granulosa cells (GCs) on human oocyte maturation in vitro, the fertilization rate, and embryo development. Methods: A total of 133 immature oocytes were retrieved and were randomly divided into two groups; oocytes that were cultured with GCs (group A) and oocytes that were cultured without GCs (group B). After in vitro maturation, only oocytes that displayed metaphase II (MII) underwent the ICSI procedure. The maturation and fertilization rates were analyzed, as well as the frequency of embryo development. Results: The mean age of the patients, their basal levels of follicle-stimulating hormone, and the number of oocytes recovered from the patients were all comparable between the two study groups. The number of oocytes that reached MII (mature oocytes) was 59 out of 70 (84.28%) in group A, compared to 41 out of 63 (65.07%) in group B (p=0.011). No significant difference between fertilization rates was found between the two study groups (p=0.702). The embryo development rate was higher in group A (33/59, 75%) than in group B (12/41, 42.85%; p=0.006). The proportion of highest-quality embryos and the blastocyst formation rate were significantly lower in group B than in group A (p=0.003 and p<0.001, respectively). Conclusion: The findings of the current study demonstrate that culturing immature human oocytes with GCs prior to ICSI improves the maturation rate and the likelihood of embryo development.

• Asian-Australasian Journal of Animal Sciences
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• 제24권2호
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• pp.190-197
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• 2011

The objective of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co-culture on developmental capacity of bovine IVM/IVF embryos and to determine whether or not melatonin acts as an antioxidant in BOEC culture and subsequent embryo development. These studies examined the effects of melatonin against NO-induced oxidative stress on cell viability, lipid peroxidation (LPO) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl-2, Caspase-3 and Bax) during BOECs culture. We also evaluated the developmental rates of bovine IVM/IVF embryos with BOEC co-culture, which were pre-treated with melatonin ($1,000\;{\mu}M$) in the presence or absence of sodium nitroprusside (SNP, $1,000\;{\mu}M$) for 24 h. Cell viability in BOECs treated with SNP (50-$2,000\;{\mu}M$) decreased while melatonin addition (1-$1,000\;{\mu}M$) increased viability in a dose-dependent manner. Cell viability in melatonin plus SNP ($1,000\;{\mu}M$) gradually recovered according to increasing melatonin addition (1-$1,000\;{\mu}M$). The LPO products were measured by thiobarbituric acid (TBA) reaction for malondialdehyde (MDA). Addition of melatonin in BOEC culture indicated a dose-dependent decrease of MDA, and in the SNP group among BOECs treated with SNP or melatonin plus SNP groups MDA was significantly increased compared with SNP plus melatonin groups (p<0.05). In expression of apoptosis or antioxidant genes detected by RT-PCR, Bcl-2 and antioxidant genes were detected in melatonin or melatonin plus SNP groups, while Caspase-3 and Bax genes were only found in the SNP group. When bovine IVM/IVF embryos were cultured for 6-7 days under the BOEC co-culture system pre-treated with melatonin in the presence or absence of SNP, the highest developmental ability to blastocysts was obtained in the $1,000\;{\mu}M$ melatonin group. These results suggest that melatonin has an anti-oxidative effect against NO-induced oxidative stress on cell viability of BOECs and on the developmental competence of bovine IVM/IVF embryo co-culture with BOEC.

• 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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• 제18권1호
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• pp.7-13
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• 1994

To verify in vivo viability of IVF-derived bovine embryos, morula and blastocysts that developed from in vitro matured and fertilized ova were transferred to the uteri of recipient cows and normal calves were produced. To produce IVF-derived bovine morula or blastocysts, ova matured and fertilized in vitro were cultured in culture medium for 7~8 days at 39$^{\circ}C$ under the humicified atmosphere of 5% CO2. Two different culture systems, a co-culture system with TCM-199 and bovine epithelial cells (BOEC) and CR1aa without somatic cell support, were compared. Cleavage rates to 2~8 cell stage and developmental rates of IVF-derived bovine embryos to blastocyst stage were not different between co-culture system (51.3 and 14.0%) and CR1aa medium (60.4 and 22.1%), respectively. Embryos were classified into three grades by embryo quality and then one or two embryos in higher quality(A and B grades) were transferred to the uterus of recipients. In this study Korean Native calf was first born after transfer of IVF-derived embryos. Total four live calves were normally developed to term from IVF-derived bovine blastocysts and one female fetus was still-born approximatedly 8 months of gestation, but there was no pregnancy after transfer of morula. Therefore, normal calves could be produced after transfer of IVF-derived bovine embryos cultured in CR1aa medium without somatic cell support. In addition, our results suggest that in transfer of IVF-derived bovine embryos blastocyst stage is better than morula.

• 대한수의학회지
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• 제36권4호
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• pp.919-927
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• 1996

In the last few years, methods for in vitro culture of early embryo stages from oocytes matured and fertilized in vitro using suitable cell culture systems have been established. But the factors affecting pregnancy rates following transfer of bovine embryos produced in vitro were not evaluated enough. So this study was performed to investigate the effects of quality and stage of embryos, parity and Corpus Luteum quality of recipients on pregnancy rates following non-surgical transfer of bovine embryos produced in vitro. Oocytes aspirated from small antral follicles of ovaries obtained at a local slaughter house were matured, fertilized with frozen-thawed semen and co-cultured for 6-7 days by utilizing co-culture system with bovine oviduct epithelial cell in vitro. After co-culture, embryos were transfered to recipients on day 7 (estrus=day 0). Recipients were monitored by ultrasonic scanning method or observation for estrus and rectal palpation after 50 days from transfer. The results of this study are follows. 1. Of the 70 recipients, 70%(49 of 70) had not showed estrus sign between day 0 and day 50, but 22.9%(16 of 70) was diagnosed not pregnant. Therefore the overall pregnancy rate of this study was 47.1%(33 of 70). 2. The pregnancy rate of recipients transfered with excellent(66.7%) and good(54.5%) embryos were higher than that of recipients transfered with fair embryos(15.8%) (p<0.05). 3. The pregnancy rate of recipients transfered with morula, compacted morula, blastocyst and expanded blastocysts were 46.2, 55.0, 62.5 and 50.0%, respectively. 4. The pregnancy rates of recipients transfered to heifer and cow were 54.5 and 55.2%, respectively. 5. The pregnancy rates of recipients with CL score I, II(66.7, 63.6%) were higher than those of recipients with CL score III (10%), (p<0.05). Success of transfer of embryos produced in vitro depends on many variables. The important factors identified in this study were the quality of embryos and the CL score of recipient animals after non-surgical transfer of embryos matured, fertilized and cultured in vitro.

• 한국수정란이식학회지
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• 제10권2호
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• pp.115-120
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• 1995

This study was carried out to investigate On in vitro fertilization, survival rate and developmental rate of rapidly frozen bovine immature oocytes. Immature oocytes cultured for 1, 12, 24, 48 hours in 20% FCS + TCM-199 medium and thereafter rapidly freezing-thawed oocytes inseminated with capacitated sperm. The immature oocytes following dehydration by 1.5M DMSO + 2.0M glycerol + 0.25M sucrose + TCM 199 media + 20% FGS were directly plunged into liquid nitrogen and thawes in 3$0^{\circ}C$ water. Rapid freezing embryos co-cultured in 20% FCS + TCM-199 media containing hormones(21U/mL PMSG, 21U /mL hGG and 1 $\mu$g /mL 17$\beta$-estradiol) and cumulus cells(1 x 105-6 cells). Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The in vitro maturation and fertilization rate of immature bovine oocytes on in vitro maturation period(1, 12, 24, 48 hrs) before rapid freezing4hawed were 57.1%, 45.7%, 37.1%, 25.7% and 40.0%, 31.4%, 20.0%, 11.4%, respectively. 2. The survival rate of immature bovine oocytes on in vitro maturation period(1, 12, 24, 48 hrs) before rapid freezing-thawed were 33.3%, 26.7%, 20.0%, and 10.0%, respectively. The survival rate of rapid freezing4hawed immature oocytes was significantly lower than that of non-freezing oocytes. 3. The survival rate of rapid freezing4hawed excellent and good bovine embryos co-cultured in 20% FCS + TCM-199 media containing hormones(PMSG, hCG, 17$\beta$-estradiol) and cumulus cells 4 to 5 hrs and 20 to 24 hrs were 35.0%, 15.0% and 25.0%, 15.0% and 40.0%, 20.0% and 30.0%, 15.0%, respectively. The survival rate of embryos co-cultured in TCM-199 media containing hormones and cumulus cells was significantly higher than that of non co-culture.

• 한국가축번식학회지
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• 제22권1호
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• pp.35-42
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• 1998

This experiment was carried out to improve in vitro development of rabbit one-cell embryos to the blastocyst stage. One-cell rabbit embryos were collected at 19\ulcorner20hr after superovulation induction and incubated at 39\ulcorner in 5% CO2 for 72hr. In order to find optimum conditions in medium that affects the rabbit embryo's development in vitro, RDH medium which mixed with RPMI1640, DMEM and Ham's F10 was compared with the previously reported mediums (Ham's F10 and RD) for embryo development and cell numbers. Three additives (BSA, taurine and glucose) were tested for the development of rabbit one-cell embryos in vitro. When the embryos were cultured in RDH medium, their development was markedly promoted as compared with Ham's F-10 or RD alone. Glucose exhibited no significant effects on embryo development and cell numbers. BSA a, pp.ared to promote transition from morula to blastocyst stage and taurine increased cell numbers of cultured embryos markedly regardless of medium. BSA and taurine together in RDH medium showed the additive effects on embryos development and cell number.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.377-383
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• 1997

The effects of embryo number and incubation volume on the development of mouse embryos were evaluated. The growth rate of two-cell mouse embryos to attached blastocyst stage and the growth rate of blastocysts to early somite stage were assessed after culture in different incubation volumes and embryo densities. Embryos were collected from ICR female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by ICR males. In experiment 1, groups of one, five, ten, twenty 2-cell embryos were cultured in 10-, 50-, 500-, 1000-${\mu}l$ drops of BWW media under mineral oil at $37^{\circ}C$ in a humidified atmosphere of 5% $CO_{2}$ and 95% air. As the incubation volume decreased, significantly (p<0.05) higher rates of embryos reached morular and blastocyst stage on day 3 and 4 culture, respectively. In experiment 2, groups of one, five, ten, twenty blastocysts were cultured in 1- and 2-ml volumes of CMRL 1066 media under same condition as in experiment 1. However the reverse was the result. Decreasing the number of embryos incubated per volume from 1 to 20 significantly (p<0.05) increased the number of blastocysts reaching the late egg cylinder (LEC) and early somite (ES) stage on day 6 and 8 culture, respectively, regardless of incubation volume. Blastocysts cultured in 2ml had higher (p<0.05) development rates to LEC and ES stage on day 6 and 8 culture, respectively, than embryos cultured in 1ml. Our results suggest that the effects of embryo number and incubation volume on the development of mouse embryos are stage specific and the shifting point was between hatching and EEC stage.