• 한국가축번식학회지
• /
• 제20권3호
• /
• pp.239-250
• /
• 1996

The objective of the present experiments were to determine whether micromanipulative and electro-stimulation conditions for blastomere survival overlapped those for oocyte activation in porcine. Eggs selected for in vitro development potential of blastomeres isolated from 4-cell embryos and oocyte activation by electrostimulation were equilibrated for 5~10 min, in 0.3M sucrose solution containing 7.5$\mu\textrm{g}$/ml cytochalasin B, and then electrostimulated for 30$\mu$sec using one pulse of 100, 120, 150 or 180 volts DC with electrodes 0.2mm apart. Single blastomeres were inserted into empty zona pellucida prior to electrostimulaticn. Then they were cultured in 20${mu}ell$ drops of fresh BECM to observe their developmental ability in vitro in a humidified incubat or at 38.5$^{\circ}C$. The results obtained from these experiments are as follows : 1. When one pulse of 100, 120, 150 or 180 volts DC for 30$\mu$sec were applied to porcine oocytes having the slit formed on zona pellucida for activation, activation rates were 65.1, 66.7, 70.7 and 91.7%, respectively. Higher activation rate was observed in 180V. 2. Infact oocytes incubated for 30 min, in 0.3M sucrose solution after electrostimulation were significantally different from control group with increasing of voltages(p<0.05). When voltages used for electrostimulation were increased, activation rates of oocytes were improved in all treatment groups. 3. When zona punctured-oocytes were only electrostimulated, or incubated in 0.3M sucrose solution for 30 min. after electrostimulation at 180 volt DC, activation rates were 90.5 and 95.5%, respectively. And activation rates of zona punctured-oocytes were significantly different from the groups for which zona pellucida was not punctured(P<0.05). 4. When single blastomeres form 4-cell transferred into empty zona pellucida were incubated for 0, 15 and 30 min. in 0.3M sucrose solution after electrostimulation using one pulse of 180 volt DC for 30 $\mu$sec, developmental rates of electrostimulated-single blastomeres to blastocyst were 72.5, 59.0 and 51.2%, respectively, and the ratio of control group developed to blastocyst were 80.0%. 5. The average cell number in electrostimulated-blastomeres developed to blastocyst were 7.9~10.8, and reduced than the cell number in diploid control ; Also cell number decreased with increasing of voltages. The results of these experiments indicate that the optimal condition for achieving in vitro developmental ability of single 4-cell blastomeres and oocyte activatin is 1 pulse, duration 30 $\mu$sec. in 180 volt, and incubation of blastomeres and oocytes in 0.3M sucrose solution after electrostimulation was not significantally different from another treatment groups. The results also show that this condition is suitable for nuclear transplantation using porcine eggs.

• Asian-Australasian Journal of Animal Sciences
• /
• 제18권1호
• /
• pp.107-112
• /
• 2005

A high environmental temperature affects the economic performance of pigs. Heat shock protein 70 (HSP70) has been reported to participate importantly in thermotolerance. This study aims to produce transgenic pigs overexpressing porcine HSP70.2, the highly inducible one of HSP70 members, and to prove the cellular thermotolerance in the primary fibroblasts from the transgenics. A recombinant plasmid in which the sequence that encodes the porcine HSP70.2 gene is fused to green fluorescence protein (GFP) was constructed under the control of cytomegalovirus (CMV) enhancer and promoter. Two transgenic pigs were produced by microinjecting pCMV-HSP70-GFP DNA into the pronucleus of fertilized eggs. Immunoblot assay revealed the varied overexpression level (6.4% and 1.4%) of HSP70-GFP in transgenic pigs. After heating at $45^{\circ}C$ for 3 h, the survival rate (78.1%) of the primary fibroblast cells from the highly expressing transgenic pig exceeded that from the non-transgenic pig (62.9%). This result showed that primary fibroblasts overexpressing HSP70-GFP confer cell thermotolerance. We suggest that transgenic pigs overexpressing HSP70 might improve their thermotolerance in summer and therefore reduce the economic loss in animal production.

• 한국가축번식학회지
• /
• 제20권1호
• /
• pp.1-8
• /
• 1996

돼지난포란의 체외성숙, 체외수정 및 체외 배양체계의 개발은 핵치환에 의한 복제동물 및 형질전환 동물생산 등과 같은 첨단생명공학연구 추진에 크게 기여할 것이다. 그러나 돼지난자의 체외수정시 다정자 침입은 큰 문제점으로 지적되고 있는데, 그 원인 및 개선책은 많은 연구에도 불구하고 정확히 밝혀져 있지 않다. 따라서 본 연구는 돼지 난포란으로부터 채취한 난자를 체외성숙 후, 체외수정시 체외수정 배양액에 난관액을 첨가하여 수정율 및 다정자 침입율을 조사하고, 또한 체외수정 후 체외 배 발달율을 조사할 목적으로 실시하였다. 수정용 배양액에 1과 5% 난관액을 첨가하였을 때 정자침입율과 수정시 난자에 침입한 정자의 평균 수가 감소하였으며, 또한 투명대에 부착된 정자의 수도 감소하였다. 난관액이 첨가된 수정용 배양액에서 정자를 1.5와 3시간 동안 전 배양을 실시하였을 때 정자의 수정능 획득과 첨체반응이 대조군에 비해 증가되었다. 그리고 1% 난관액이 포함된 배양액에서 체외수정된 난자의 배반포까지 발달율은 18.9%로 대조구의 12.4%보다 유의성 있게 높았다. 이러한 연구결과는 난관 분비액의 어떤 인자가 정자의 첨체반응을 유도해서 정자침입율 및 다정자 침입을 감소시키고, 이어 배발달율을 증가시킨다고 사료된다.

• 농업과학연구
• /
• 제23권2호
• /
• pp.212-218
• /
• 1996

본 연구는 체외에서 성숙 및 수정된 돼지 체외 수정란을 두 종류의 서로 다른 배양액에 단층난구 세포와 공배양하였을 경우 배발달에 미치는 영향을 조사하기 위하여 실시하였다 본 연구에서 얻은 결과를 요약하면 다음과 같다. 1. 단층난구세포와 함께 초기체외수정란을 Ham's F-10에서 배양한 결과 2-, 4-, 8~16-세포기, 상실배/배반포기로 발달하는 비율은 각각 48.7%, 40.5%, 34.8% 및 17.0% 였으며, TCM-HEPES에서는 각각 48.3%, 35.6 %, 22.1% 및 13.4%였다. 2. 단층난구세포와 공배양을 하지 않고 단순하게 배양액으로만 배양하였을 때 상실배/배반포기까지의 발달률은 0.0~1.0%로서 극히 저조하였다. 특히, 대부분의 수정란이 4-세포기 단계에서 발달이 정지되었다. 3. 돼지 체외수정란을 단층난구세포와 공배양하였을 때 단순배양한 결과보다 유의 적으로 높은 상실배/배반포기의 발달률을 나타냈으며 (P<0.05), 두 배양액간에는 유의차가 인정되지 않았다.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2004년도 춘계학술발표대회
• /
• pp.217-217
• /
• 2004

The present study was conducted to investigate the developmental competency between male- and female-somatic cell derived nuclear-transferred porcine embryos, and the productive and survival efficiency of cloned male and female piglets. The potential of eggs receiving somatic cells to develop into blastocysts was not different among donor cells of different origins. (omitted)

• 한국가축번식학회지
• /
• 제17권2호
• /
• pp.103-111
• /
• 1993

These experiments were undertaken to establish the optimal culture systems for in vitro maturation, fertilization and subsequently embryonic development of porcine immature follicular oocytes isolated from the ovary of slaughtered pigs. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles (3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated (39$^{\circ}C$, 5% CO2 in air) for 42hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were prepared forfertilizaing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${mu}ell$ of capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the culture media. The fertilization rates of in vitro matured follicular oocytes cultured in B. O., TCM-HEPES, m-KRB, and TALP-II media were 61.3%, 83.0%, 88.9% and 89.2%, respectively. In addition, the polyspermy rates were 60.7%, 66.5%, 53.8%, and 43.9%, respectively. These data indicated that the highest of fertilization and the lowest of polyspermy rate was shown in TALP-II medium. Spermatozoa capacitated by caffeine, heparin, and percoll density gradient treatment in the 4 different media, the fertilization rates were 33.0~57.2%, 39.9~90.2%, and 52.6~92.8%, respectively, showing the lowest rate in caffeine treatment. The development rate of follicular oocytes, fertilized with the spermatozoa capacitated by caffeine, heparin, and percoll gradient in the TALP-II medium, upto 2 to 4-cell stages were 32.6%, 74.5% and 70.9%, respectively. Finally, fertilization rates of follicular oocytes cultured with follicular fluid containing medium from 10 to 100% were 61.2~94.1% and the rates (90~94%) with 10~20% follicular fluids were significantly higher than those (85.3%) of cultured in the media without follicular fluid. In addition, the rates of pronucleus formation were also higher in follicular fluid treated group (73.1~83.0%) than those (64.7%) of oocytes cultured without follicular fluid. The highest fertilization and pronucleus formation rates was found in oocytes cultured with 10% follicular fluid. These results suggest that the addition of heparin or percoll density gradient method is better capacitation method. Furthermore, the addition of porcine follicular fluid to the fertilization medium may improve the fertilization rates and formation of pronucleus.

• Reproductive and Developmental Biology
• /
• 제30권2호
• /
• pp.125-133
• /
• 2006

This study was conducted to detect the apoptosis incidence in blastocysts and to compare the abundance of Bax, Bcl2L1, VEGF and FGFR2 in in vitro fertilized (IVF), parthenogenetic (PAT) and nuclear transfer (NT) embryos. Oocytes matured for 40 hr were enucleated and reconstructed with confluenced fetal fibroblasts (FFs) derived from a ${\sim}45$ day fetus. Reconstructed eggs were then fused with 2 DC pulses (2.0 kV/cm, $30{\mu}sec$) and cultured with $7.5{\mu}g/ml$ cytochalasin B for 3 hr. Parthenotes (PAT) were produced with the same electric strength and culture for NT eggs. The embryos were cultured in NCSU-23 medium at $39^{\circ}C,\;5%\;CO_2,\;5%\l;O_2$ in air. In 3 runs, set of 10 embryos at the 4-cell to blastocyst stages were used to extract total RNA for analyzing the gene expression patterns of pro-apoptotic (Bax), anti-apoptotic (Bcl2L1), vasculogenesis (VEGF), implantation (FGFR2III) using real-time quantitative PCR. Cleavage and blastocyst rates were significantly higher (P<0.05) in IVF and PAT ($79.3{\pm}8.5\;and\;25.5{\pm}6.1,\;and\;85.0{\pm}6.4\;and\;38.6{\pm}5.5$, respectively)than NT counterparts ($65.1{\pm}5.2\;and\;15.6{\pm}3.0$, respectively). Significantly higher (P<0.05) total cells were observed in IVF controls and PAT ($34.7{\pm}5.8\;and\;38.1{\pm}4.1$) than NT embryos ($24.8{\pm}3.2$). Apoptosis index was significantly lower (P<0.05) in IVF than NT embryos. The Relative abundances (RA) of Bax and VEGF were significantly higher (P<0.05) at blastocyst stage in NT than IVF control. The RA of Bcl2L1 and FGFR2III were significantly higher (P<0.05) at blastocyst stage in IVF than NT. The present study observed the abnormal gene expressions in NT embryos at various developmental stages, suggesting certain clues to find out the cause of the low efficiency of NT to term.

• Reproductive and Developmental Biology
• /
• 제32권2호
• /
• pp.135-140
• /
• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• 한국발생생물학회지:발생과생식
• /
• 제15권1호
• /
• pp.31-39
• /
• 2011

Change in intracellular $Ca^{2+}$-concentration ($[Ca^{2+}]_i$) is an essential event for egg activation and further development. $Ca^{2+}$ ion is originated from intracellular $Ca^{2+}$-store via inositol 1,4,5-triphosphate receptor and/or $Ca^{2+}$ influx via $Ca^{2+}$ channel. This study was performed to investigate whether changes in $Ca^{2+}$/calmodulin dependent protein kinase II (CaM KII) activity affect $Ca^{2+}$ influx during artificial egg activation with ethanol using $Ca^{2+}$ monitoring system and whole-cell patch clamp technique. Under $Ca^{2+}$ ion-omitted condition, $Ca^{2+}$-oscillation was stopped within 30 min post microinjection of porcine sperm factor, and ethanol-induced $Ca^{2+}$ increase was reduced. To investigate the role of CaM KII known as an integrator of $Ca^{2+}$- oscillation during mammalian egg fertilization, CaM KII activity was tested with a specific inhibitor KN-93. In the eggs treated with KN-93, ethanol failed to induce egg activation. In addition, KN-93 inhibited inward $Ca^{2+}$ current ($I_{Ca}$) in a time-dependent manner in whole-cell configuration. Immunostaining data showed that the voltage-dependent $Ca^{2+}$ channels were distributed along the plasma membrane of mouse egg and 2-cell embryo. From these results, we suggest that $Ca^{2+}$ influx during fertilization might be controlled by CaM KII activity.

• 한국가축번식학회지
• /
• 제22권4호
• /
• pp.395-403
• /
• 1998

본 연구에서는 돼지난자내 원형정자 주입후 수정율과 체외 배 발달을 조사하였다. 원형정자 주입 2시간 전에 인위적 전기자극을 주고 원형정자를 주입했을 때 난자들의 수정율이 주입 직후에 전기자극을 준 것들과 전기 자극을 주지 않은 것들보다 수정율이 높았다. 원형정자와 원형 정자핵을 각각 주입 한 후 전핵 형성율과 전핵 이동율을 조사하였으나 유의차를 발견할 수 없었다. 전핵의 형성과 이동중 미세소관의 움직임을 간접형광면역법 및 공춧점 현미경을 사용하여 조사해본 결과, 난활성 직후 난자의 표층에서 미세소관이 발생해서 이것에 의해 웅성 및 자성전핵이 난자 중심부로 이전됨을 볼 수 있었다. 원형정자와 원형정자핵 주입후 배양 6일째에 각각 25% 와 27%의 배반포로 형성되었고, 8 일째 형성된 배반포를 염색하여 관찰한 철과 세포수가 각각 평균 87에서 99개가 형성되었음을 알 수 있었다. 이러한 결과는 체외 성숙된 돼지 난자 내에 원형정자 혹은 원형정자 핵을 미세 주입하는 방법에 의해 정상척인 배발달을 하는 돼지 수정란 생산이 가능한 것을 보여주는 것이다.