• Reproductive and Developmental Biology
• /
• 제33권3호
• /
• pp.157-162
• /
• 2009

This study was performed to comprehend the developmental characteristics of cloned embryos knocked out (KO) of $\alpha$-1,3-galactosyltransferase (GalT) gene. Immature oocytes were collected and cultured for 40 hrs (1-step) or 20hrs (with hormone) + 20hrs (without hormone) (2-step). The embryos transferred with miniature pig ear fibroblast cell were used as control. The reconstructed embryos were cultured in PZM-3 with 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. To determine the quality of the blstocysts, TUNEL and quantitative realtime RT-PCR were performed. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle. The maturation rate was significantly higher in 2-step method than that of 1-step (p<0.05). The blastocyst development of GalT KO embryos was significantly lower than that of normal cloned embryos (p<0.05). The total and apoptotic cell number of GalT KO blastocysts was not different statistically from control. The relative abundance of Bax-$\alpha$/Bcl-xl ratio was significantly higher in both cloned blastocysts than that of in vivo blastocysts (p<0.05). Taken together, it can be postulated that the lower developmental potential and higher expression of apoptosis related genes in GalT KO SCNT embryos might be a cause of a low efficiency of GalT KO cloned miniature pig production.

• Asian-Australasian Journal of Animal Sciences
• /
• 제24권1호
• /
• pp.37-44
• /
• 2011

Parthenogenetic embryonic stem (pES) cells could provide a valuable model for research into genomic imprinting and X-linked diseases. In this study, pES cell lines were established from oocytes of hybrid offspring of Kunming and 129/Sv mice, and pluripotency of pES cells was evaluated. The pES cells maintained in the undifferentiated state for more than 50 passages had normal karyotypes with XX sex chromosomes and exhibited high activities of alkaline phosphatase (AKP) and telomerase. Meanwhile, these cells expressed ES cell molecular markers SSEA-1, Oct-4, Nanog, and GDF3 but not SSEA-3 detected by immunohistochemistry and RT-PCR. The pES cells could be differentiated into various types of cells from three germ layers in vitro by analysis of embryoid bodies (EBs) with immunohistochemistry and RT-PCR, and in vivo by observation of pES cell-derived teratoma sections. Therefore, the established pES cell lines contained all features of mouse ES cells. This work provides a new strategy for isolating pES cells from Kunming mice, and the pES cell lines could be applied as the cell model in research into genomic imprinting and epigenetic regulation of Kunming mice.

• 한국수정란이식학회지
• /
• 제30권3호
• /
• pp.225-228
• /
• 2015

Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

• Clinical and Experimental Reproductive Medicine
• /
• 제36권3호
• /
• pp.187-197
• /
• 2009

목 적: 단백질 인산화는 세포신호전달에 매우 중요한 현상으로서, 수많은 조절인자들이 난자성숙에 관여하게 된다. 그러나 이들 중에서 어떤 단백질이 인산화되어 난자성숙을 조절하는지는 잘 알려져 있지 않다. 따라서 체세포의 신호전달과정에서 인산화를 통해 중요한 기능을 한다고 알려져 있는 일곱 가지 단백질들이 생쥐의 난자성숙과정에서 어떻게 인산화 되고 있는지 알아보고자 한 개 샘플에서 일곱 개의 변화를 한꺼번에 측정할 수 있는 bead-based multiplex phosphorylation assay를 이용하여 본 연구를 수행하였다. 연구방법: ICR 생쥐에 PMSG를 주사하고 46시간 후에 cumulus-oocyte complex (COCs) 형태로 미성숙 난자를 채취한 후 체외배양 하면서, 배양 2시간 후에 GVBD를, 배양 8시간 후에 MI을, 배양 16시간 후에 MII 단계의 난자를 얻었고 체내에서 배란한 MII 단계의 난자는 수란관에서 얻었다. 각 단계의 난자를 100개씩 모아서 mitogen-activated protein kinase (MAPK)에 속하는 세가지 단백질인 ERK1/2, JNK, p38 MAPK와 Akt, GSK-$3{\alpha}/{\beta}$, $I{\kapa}B{\alpha}$, STAT3 등 총 일곱 단백질의 인산화를 Bio-Plex System을 이용하여 같은 시료에서 동시에 측정하였으며 세 번의 반복실험을 통하여 얻어진 결과를 통계적으로 분석하였다. 결 과: 생쥐의 난자성숙과정에서 측정된 일곱 가지 단백질 중에서 인산화가 현저히 증가하는 단백질로는 ERK1/2, JNK, p38 MAPK와 STAT3로서 미성숙 난자에 비해서 3배에서 20배까지 인산화되는 결과를 보였다. 반면에 GSK-$3{\alpha}/{\beta}$, $I{\kappa}B{\alpha}$의 인산화의 변화는 미약하였으며, Akt의 경우에는 변화가 전혀 없었다. 난자성숙 과정에서 분석 대상 단백질들의 인산화는 GVBD 단계에서 활성화되기 시작하여 MI에서 현저히 높게 증가하며 MII까지 높게 유지되었다. 결 론: 본 연구는 난자성숙과정에서 일곱 가지 단백질의 인산화를 동시에 측정한 최초의 보고로서 이 방법은 난자와 같이 적은 양의 시료에서의 여러 개의 단백질 인산화를 동시에 분석하는데 유용할 것으로 생각된다. 본 연구결과, 세 가지 MAPK 단백질인 ERK1/2, JNK, p38 MAPK 외에도 STAT3가 난자성숙에 있어서 매우 중요한 조절자로 생각되었다. 또한 Akt의 473번 serine기의 인산화는 난자성숙에 관여하지 않음을 알 수 있었다.

• 한국수정란이식학회지
• /
• 제19권2호
• /
• pp.133-145
• /
• 2004

난자의 체외성숙 및 체외배양에는 일반적으로 동물의 혈청을 사용하고 있다. 그러나, 채취한 소의 상태에 따라서 혈청의 질에 차이가 있어 실험데이터가 일정하지 않을 수 있고, 그것으로부터 바이러스, 세균, 마이코플라즈마 등에 오염될 가능성이 있다. 따라서, 본 실험에서는 완전 무 혈청 배양액에서 난자의 성숙, 배 발생율, 세포 수, 동결성을 검토하였다. 다음으로, 근래 혈청배지로 생산한 체외 배양 수정란은 과체중의 산자 생산, 초기 산자 사망률, caesarean section, dystocia 같은 발병으로 문제가 지적되고 있다. 그러나 무 혈청 배지로 생산한 체외 배양 수정란은 이러한 현상을 개선할 수 있다는 보고가 있어 본 실험에서는 완전 무혈청 배양액에서 생산된 동결란과 신선란을 젖소에 이식하여 그 결과를 검토하였다. 무혈청 배양액에는 에너지원과 세포성장인자가 첨가된 IVD101과 IVMD101, 대조군인 혈청 배양액에는 TCM199 + 10% FBS가 사용되었다. IVMD101에 의해 체외성숙이 이루어 졌고, 혈청이 첨가된 TCM199 + 10% FBS에서는 12.4% 배 발생율을 보인데 반해, 무혈청 배양액 IVD101과 IVMD101이 각 32.4%, 34.5%로 훨씬 높은 배 발생율을 보였다. 더욱이, 세포 수에 있어서도 무혈청 배양액에서 발생된 배반포의 세포수가 혈청이 첨가된 배양액에서 발생된 배반포의 세포수보다 월등하였으며, 이는 체내란과 비슷한 세포 수를 보이고 있다. 수정란의 내동성을 보면, 융해하고 24시간 배양기 정치 후에 IVD101과 IVMD101은 94.5%, 95.8%의 생존율을 보인 반면 TCM199 + 10% FBS는 52.5%에 불과하다. 융해하고 72시간후 탈출 배반포율이 IVD101과 IVMD101는 78.4%, 83.7%였는데 반해 TCM199 + 10% FBS는 32.0%였다. 마지막으로, 무혈청 배양액에서 발생한 동결란과 신선란을 젖소에 이식한 후에 임신율에 있어서는 유의적 차이는 없었지만, 무혈청 배지에서 생산된 수정란의 내동성의 탁월함이 증명되었다. 이러한 결과들로 종합해 보면, 무혈청 배양액 (IVD101, IVMD101)에서 발생된 수정란이 혈청 배양액에서 발생된 수정란보다 배발생율, 세포수, 동결성, 임신율에 있어서 우수함을 알 수 있었다. 이러한 무혈청 배양액은 배발생 과정의 연구뿐만 아니라 수정란 이식을 위한 고품질의 체외 수정란의 대량 생산, 복제, 형질전환 동물 생산 등에도 유익할 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
• /
• 제23권1호
• /
• pp.1-6
• /
• 1996

There are a number of problems during the process of culture in vitro on fertilization and embryo development compared to those on in vivo counterparts. And the platelet activating factor (PAF), which is found not only in mammalian spermatozoa but also preembryos, is implicated on reproductive process. To improve the environment of culture on in vitro fertilization and embryo development, coculture using salpingeal epithelial cells has been considered to accept the better result on pregnancy rate. This study was designed to determine if two different culture systems, coculture alone and PAF treated coculture, are positive or negative influence on process of in vitro fertilization and embryo culture in mouse. The cell cleavage rate reached to 2-4 cell stage at 24 hours of culture is 56.81% (50/88) and 48.21%(54/112) respectively, in PAF treated group which is added PAF on coculture and in coculture group. But the rate of cells cleavage was similar in both group after 48 hours of culture. The rate of unfertilization after insemination of oocytes was higher in coculture group(55..53%) than in PAF treated group(42.37%). And in assessment of undeveped embryos, the rate of equalized cell block was similar on both, coculture alone (35.3%)and PAF treated coculture(35.5%). while unequalized cell block was higher rate in PAF treated coculture(19.4%) than coculture alone (11.8%). But the rate of cytoplasmic degeneration of undeveloped embryos was significantly higher in PAF treated coculture than coculture alone. In conclusion, we have observed that PAF treated coculture is superior in the rates of in vitro fertilization and early embryo cell cleavage compared to those in coculture alone, but there is no difference on the rates of embryo develpments, cell degeneration, cell quality in both PAF treated coculture and coculture alone when the embryo cells were continuosly cultured for 48 hours or more.

• Reproductive and Developmental Biology
• /
• 제34권3호
• /
• pp.127-134
• /
• 2010

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 ($\geq$ 2-cell) embryos were cultured in 0 (control), 1, 10 and $20\;{\mu}M$ flavonoid for 6 days. In the results, in vitro development rate was the highest in $10\;{\mu}M$ flavonoid group (57.1%) among treatment groups (control, 49.5%; $1\;{\mu}M$, 54.2%; $20\;{\mu}M$, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in $10\;{\mu}M$ flavonoid group than other groups. We found that $10\;{\mu}M$ flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in $10\;{\mu}M$ flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, $10\;{\mu}M$ flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in $10\;{\mu}M$ flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

• Clinical and Experimental Reproductive Medicine
• /
• 제35권2호
• /
• pp.111-118
• /
• 2008

연구방법: 미성숙 백서 난소의 과배란 유도를 위해 PMSG를 주사하고, 배란을 위해서 hCG를 주입하였다. RGS-2의 유전자 발현양상을 조사하기 위하여는 Northern blot 분석과 in situ hybridization 분석을 시행하였다. 결 과: 미성숙 백서에 성선자극호르몬인 PMSG를 복강내 주사했을 때 RGS-2 mRNA 발현에 영향을 미치지 않음을 Northern blot analysis로 확인할 수 있었으나, hCG를 주입했을 때는 1시간에서 3시간 내에 발현이 증가됨을 알 수 있었다. In situ hybridization으로 살펴본 RGS-2 mRNA의 발현세포는 난포의 크기에 관계없이 난자였으나, hCG로 처리한 후에는 배란 전 난포와 성장중인 난포의 과립막 세포이었다. 그러나, RGS-2 단백의 발현은 hCG 처치와 관계없이 난포막 세포이었다. 상기 생체 실험과 마찬가지로 시험관에서도 배란 전 난포의 과립막 세포에 대한 LH 처리는 RGS-2 유전자 발현을 1시간 내에 촉진하였다. 또한, 성선자극호르몬 분비호르몬 2 길항제도 이러한 LH의 촉진작용을 증진시켰다. 결 론: 본 연구로 배란 전 과립막 세포에서 성선자극호르몬인 LH/hCG와 성선자극호르몬 분비호르몬 길항제에 의해 RGS-2의 발현이 증진되는 양상으로 보아 RGS-2가 배란과정 동안에 Gq protein 신호전달을 조절할 것으로 추정된다.

• 한국수정란이식학회지
• /
• 제31권3호
• /
• pp.221-226
• /
• 2016

The aim of the present study was to assess the embryo development and survivability of post-thawed bovine embryos produced in vitro by addition of cysteine. The rates of metaphase II formation were not differed significantly among three groups(TCM199 73.8%, TCM199 with 0.3% cysteine 76.9%, TCM199 with 0.5% cysteine 83.8%, respectively). No difference of cleavage rate(70.6~74.6%) was seen among three culture medium(TCM199 70.6%, CR1aa 71.3%, SOF 74.6%) with 0.5M cysteine. however, Significantly(P<0.05) higher development rate into blastocyst stage by 0.5M cysteine addition was obtained in SOF medium(35.6%) than in TCM199(27.6%) or CR1aa(26.6%), however no significant differences in the cleavage rates were among three culture medium. After frozen the blastocysts cultured with 0.5M cysteine, The re-expansion rates were 61.3%~86.4% among groups, and hatching rates were 26.3%~46.9% among groups, the rates of re-expansion and hatching were significantly(P<0.05) higher in SOF medium(86.4% and 46.9%) than those in TCM199(61.3% and 26.3%) and CR1aa medium(87.1 and 44.4%). After thawing, the blastocyst re-expansion rate was significantly(P<0.05) higher in in vivo (87.1%) and in vitro (70.3%) embryos. In conclusion, our results demonstrate that supplementation of IVM and IVC medium with 0.5M cysteine improved the quality of in vitro production embryo and post- thawed embryo. Future studies comparing these media systems in well-designed trials should be performed.

• 한국수정란이식학회지
• /
• 제23권2호
• /
• pp.101-108
• /
• 2008

This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-${\alpha}$, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax-${\alpha}$ was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-${\alpha}$/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.