• Asian-Australasian Journal of Animal Sciences
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• 제28권11호
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• pp.1565-1572
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• 2015

Porcine embryonic stem cells (pESCs) have become an advantageous experimental tool for developing therapeutic applications and producing transgenic animals. However, despite numerous reports of putative pESC lines, deriving validated pESC lines from embryos produced in vitro remains difficult. Here, we report that embryo aggregation was useful for deriving pESCs from in vitro-produced embryos. Blastocysts derived from embryo aggregation formed a larger number of colonies and maintained cell culture stability. Our derived cell lines demonstrated expression of pluripotent markers (alkaline phosphatase, Oct4, Sox2, and Nanog), an ability to form embryoid bodies, and the capacity to differentiate into the three germ layers. A cytogenetic analysis of these cells revealed that all lines derived from aggregated blastocysts had normal female and male karyotypes. These results demonstrate that embryo aggregation could be a useful technique to improve the efficiency of deriving ESCs from in vitro-fertilized pig embryos, studying early development, and deriving pluripotent ESCs in vitro in other mammals.

• 한국수정란이식학회지
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• 제10권2호
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• pp.121-129
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• 1995

To examine the critical effect of oxygen concentration on embryonic development, in vitro fertilized embryos were cultured in media(TCM199 vs. SOF) supplemented sera(1O% FCS vs. 10% HS) with and without bovine oviduct epithelial cells under two gas atmosphere (5% $CO_2$ in air vs. 5% $CO_2$, 5% $O_2$, 90% $N_2$). Oocytes, obtained from abattoir ovaries, were matured in EGF containing TCM199 medium co-cultured with BOEC for 24 hours, followed by exposure to frozen-thawed, heparin4reated spermatozoa in TALP for 30 hours. And then early embryos(1~2 cell) were cultured in both TCM199 and SOF supplemented with 10% FCS or 10% RS under 5% $CO_2$ in air or 5% COi, 5% $O_2$, 90% $N_2$. Development to morulae and blastocysts was recorded on days 7, after the start of in vitro fertilization. The developmental rates of in vitro fertilized embryos to morulae and blastocysts cultured in SOF with BOEC under 5% $CO_2$, 5% $O_2$, 90% $N_2$(24.4%) were significantly(p<0.05) higher than cultured in SOF with BOEC under 5% $CO_2$ in air(14.1%) at seven days after in vitro fertilization. When early bovine embryos were cultured in TCM 199 and SOF under two different gas atmosphere, there were no significant differences in the developmental rates to morulae and blastocysts between supplements of 10% FCS and 10% HS. The rates of development to morulae and blastocysts were significantly(p<0.01) higher in TCM 199 with BOEC(24.7%) than TCM199 without BOEC(10.9%) under 5% $CO_2$ in air, otherwise SOF without BOEC(36.4%) were significantly (p<0.05) higher than in SOF with BOEC (24.4%) under 5% $CO_2$, 5% $O_2$, 90% $N_2$. In summary, these experiments have proved that the culture system in SOF supplemented 10% ES is effective on in vitro development of early bovine embryos under 5% $CO_2$, 5% $O_2$, 90% $N_2$. In addition, it is effective to development of bovine embryos that TCM 199 should be co-cultured with BOEC and SOF should be cultured without somatic cells under two different gas atmosphere.

• Asian-Australasian Journal of Animal Sciences
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• 제23권6호
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• pp.693-699
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• 2010

Imprinted genes are essential for fetal development, growth regulation, and postnatal behavior. However, little is known about imprinted genes in livestock. We hypothesized that certain putatively imprinted genes affected normal peri-implantation development such as embryo elongation, initial placental development, and preparation of implantation. The objective of the present study was to investigate the mRNA expression patterns of several putatively imprinted genes during the porcine peri-implantation stages from day 6 to day 21 of gestation. Imprinted genes were selected both maternally (Dlk1, IGF2, Ndn, and Sgce) and paternally (IGF2r, H19, Gnas and Xist). Here, we report that the maternally imprinted gene IGF2 was expressed from day 6 (Blastocyst stage), but Dlk1, Ndn, and Sgce were not expressed in this stage. These genes were first expressed between days 12 and day 14. All the maternally imprinted genes studied showed significantly high expression patterns from day 18 of embryo development. In contrast, paternally imprinted genes IGF2r, H19, Gnas, and Xist were first expressed from day 6 of embryo development (BL). Our data demonstrated that the expression of H19 and Gnas genes was significantly increased from day 14 of the embryo developmental stage, while IGF2r and Xist only showed high expression after day 21. This study is the first to show that the putatively imprinted genes were stage-specific during porcine embryonic development. These results demonstrate that the genes studied may exert important effects on embryo implantation and fetal development.

• 한국동물학회지
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• 제33권3호
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• pp.365-372
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• 1990

Transgenic 양서류를 만들기 위한 연구의 일환으로 bacterial $\beta$-galactosidase 유전자를 cytopalsmic actin promoter에 연결시킨 plasmid를 xenopus 수정란에 미세주입하여 외부 DNA의 발현을 조사하였다. $\beta$-gal DNA를 20nl당 1ng에서 2ng의 농도로 미세주입하는 경우, 이 농도는 배발생에 크게 영향을 미치지 않는 것으로 나타났다. 또한 유전자 산물인 $\beta$-galactosidase는 양서류의 모든 배엽성 세포에서 발현 가능하고, 정상적인 활성을 나타내므로 외부 DNA의 발현여부를 in situ 상태에서 판명할 수 있었다. 주입된 외부 DNA는 낭배기 시기에 최초로 발현되고, 적어도 올챙이 시기까지 유지 및 발현 가능하며, 초기 발생동안 extrachromosomal 상태에서 발현되는 것으로 나타났다. 그러나 발현의 정도는 주입된 개체는 물론 매 실험마다 차이를 보였고, 또한 기질인 X-Gal에 반응하는 부위가 전체 배에 분포하는 겅우는 거의 없으며, 일정 부위에 한정되어 있음이 관찰되었다. 이는 세포막을 통해서 DNA가 다른 할구로 이동하지 못하는 사실에 비추어 미세주입된 DNA가 난할시 각 할구로 균등하게 분포되지 못하고 국부적으로 나뉘어 들어가기 때문인 것으로 사료된다.

• 생태와환경
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• 제44권1호
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• pp.1-8
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• 2011

미꾸리과 어류에 속하는 우리나라 고유종인 북방종개를 강원도 고성군 북천에서 채집하여 난발생과정과 초기 생활사를 연구하였다. 채집된 성숙한 북방종개를 호르몬(LHRH-a, HCG) 주사하여 채란하고 건식법으로 수정 시켰다. 수정란은 원형이었고 엷은 흰색을 띠는 분리침성란이며, 난경은 $1.09{\pm}0.04\;mm$ (n=20) 이었다. 수온 $21.0{\sim}24.0^{\circ}C$에서 수정 후 48시간 전후하여 부화하였으며, 부화자어의 크기는 전장 $2.87{\pm}0.05\;mm$ (n=20) 이었다. 부화 4 일 후에는 전장 $6.86{\pm}0.10\;mm$ (n=10)로 성장하였고, 난황이 거의 흡수되고 입과 항문이 열렸다. 부화 14 일 후에는 전장 $10.71{\pm}0.34\;mm$ (n=10)로 자라고 대부분의 지느러미 기조가 출현하였으며 반문이 나타났다. 부화 26일 후에는 전장 $14.88{\pm}0.45\;mm$ (n=10)로 자라고 모든 지느러미의 기조수가 정수에 도달하여 치어기로 이행되었다. 부화 80일 후에는 전장 $33.3{\pm}1.25\;mm$ (n=10)로 성장하였으며, 반문의 모양과 외부형태가 성어와 유사하였다. 북방종개의 배 발생 및 초기생활사 특정은 다른 미꾸리과 어류들과 큰 차이를 보이지 않았으며, 추후 고유종의 복원연구에 기초자료를 제공하였다.

• 한국발생생물학회지:발생과생식
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• 제12권3호
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• pp.283-288
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• 2008

이 연구는 2003년 4월부터 5월까지 전라남도 보성군 겸백면 율어리 소재의 보성강 중류에서 투망과 족대를 이용하여 채집된 모래무지 어미들을 전남대학교 해양기술학부 자원생물실험실로 운반하여 실내 사육하면서 난발생 및 자치어 형태발달을 관찰하였다. 모래무지의 수정란은 구형의 침성부착란으로 난경은 $1.98{\pm}0.19mm$(n=50)였고, 반투명하였다. 부화에 소요되는 시간은 수정 후 164시간부터였고, 이 때 근절수가 31$\sim$32개였으며, 배체의 움직임이 활발하였으며, 머리부터 난막을 뚫고 부화하기 시작하였다. 부화 직후의 자어는 전장이 $4.61{\pm}0.83mm$(n=10)로 난황은 아직 흡수하지 않은 상태였으며, 입과 항문은 열려 있지 않았다. 등쪽과 배쪽, 꼬리쪽에 별모양과 점모양의 흑색소포가 산재하였으며, 눈에는 색소포가 진하게 착색되어 있었다. 부화 후 42일째는 전장 $16.22{\pm}0.65mm$(n=10)로 흑색소포가 두부와 등쪽과 체측면에 따라 짙게 산재하였고, 주둥이 양쪽에 수염이 나타났으며, 체형이나 반문이 성어와 완전히 닮아 있어 치어기로 이행하였다.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.281-285
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• 2011

In the present study, we identified differentially methylated region (DMR) upstream of Dnmt1o and Dnmt1s gene in early porcine embryos. Porcine Dnmt1o had at least one DMR which was located between -530 bp to -30 bp upstream from transcription start site of the Dnmt1o gene. DNA methylation analyses of Dnmt1o revealed the DMR to be hypomethylated in oocytes, whereas it was highly methylated in sperm. Moreover, the DMR upstream of Dnmt1o was gradually hypermethylated from oocytes to two cells and dramatically changed in the methylation pattern from four cells to BL stages in an in vivo. In an IVF, the methylation status in the DMR upstream of Dnmt1o was hypermethylated from one cell to eight cells, but demethylated at the Morula and BL stages, indicating that the DNA methylation pattern in the Dnmt1o upstream ultimately changed from stage to stage before the implantation. Next, to elucidate whether DNA methylation status of Dnmt1s upstream is stage-by-stage changed in during porcine early development, we analyzed the dynamics of the DNA methylation status of the Dnmt1s locus in germ cell, or one cell to BL cells. The Dnmt1s upstream was highly methylated in one and eight cells, while less methylated in two, four, morula, and BL cells. Taken together, our data demonstrated that DNA methylation and demethylation events in upstream of Dnmt1o/Dnmt1s during early porcine embryos dramatically occurred, and this change may contribute to the maintenance of genomewide DNA methylation in early embryonic development.

• 한국가축번식학회지
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• 제20권2호
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• pp.171-177
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• 1996

This study was designed to evaluate the efficacy of antioxidants and amino acid with buffalo rat liver cell(BRLC), bovine oviductal epithelial cell(BOEC) and STOC monolayers in supporting the development of in vitro matured(IVM) and in vitro fertilized(IVF) bovine oocytes. Bovine embryos developed to the 2~8 cell stage after in vitro fertilization were cultured for 5 to 6 days at 39$^{\circ}C$ in CR1aa containing antioxidants and amino acids with various somatic cells. Embryo development was examined and cell numbers of blastocysts were counted by fluorescence staining method. In experiment 1, the proportion of embryos that reached the blastocyst stage in control, catalase(250U), SOD(600U), glutathione(100$\mu$M) and taurine(2.5mM) with BRLC were 11.4, 8, 0, 16.7 and 43.4 respectively. Taurine(2.5mM) with BRLC group was significantly the highest among treatments(P<0.05). In experiment 2, in vitro development rate into blastocyst in control, catalase(250U), SOD(600U), glutathione(100$\mu$M) and taurine(2.5mM) with BOEC were 15.8, 23.5, 22.8, 28.6 and 56.9 respectively. In experiment 3, embryonic development in all treatments as control, catalase(250U), SOD(600U), glutathione(100$\mu$M) and taurine(2.5mM) added to CR1aa with STO cells were 23.5, 24.5, 17.0, 28.8 and 50.0 blastocysts. These results show that antioxidants and amino acids with somatic cells can provide a significant benefit for coculture of early bovine embryos derived from IVM and IVF.

• 한국응용곤충학회지
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• 제24권3호
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• pp.141-149
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• 1985

담배나방(Heliothis assulta Guenee)의 배자 발생을 알아보기 위하여 실내에서 우화시킨 성충의 난(卵)을 이용하여 온도에 따른 배자발생기간을 조사하였으며, $25^{\circ}C$에서의 조직분화 및 기관형성과정을 관찰하여 다음과 같은 결과를 얻었다. 1. 난(卵)은 적도직경 0.53mm, 높이 0.56mm인 구형이며, 표면에는 정방형(正方形)의 요철(凹凸)무늬가 있고, 난(卵)의 정부(頂部)에는 1개의 micropyle이 있었다. 2. 담배나방의 배자발생기간은 20, 25, 30, $35^{\circ}C$에서 각각 147, 81, 61, 67시간이 있으며 $35^{\circ}C$에서의 배자기 치사율이 크게 증가하였다. 3. 난(卵)의 외형(外形)은 발생이 진행됨에 따라 색깔 및 무늬의 변화가 뚜렷하였으며 발생시간에따라 특징적이었다. 4. $25^{\circ}C$에서는 배자발생이 시작된 후 14시간이 경과하면 기관형성이 일어나며, 44시간 경과 후에는 소화기관 형성이 완성되고, 80시간 후에 부화하였다. 5. 발생 초기에 난(卵)의 장축을 따라 형성된 배자는 발생시작 24시간 경과 후에는 $90^{\circ}$ 회전하고 , 이어서 배의 장축을 중심으로 $180^{\circ}$ 회전하며, 머리부분이 micropyle을 향하여 부화시까지 고정되었다.

• 한국발생생물학회지:발생과생식
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• 제16권4호
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• pp.363-370
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• 2012

The outer dense fiber 2 (ODF2) protein is an important component of sperm tail outer dense fiber and localizes at the centrosome. It has been reported that the RO072 ES cell derived homozygote knock out of ODF2 results in an embryonic lethal phenotype, and XL169 ES cell derived heterozygote knock out causes severe defects in sperm tail development. The ODF2s splicing variant, Cenexin1, possesses a C-terminal extension, and the phosphorylation of serine 796 residue in an extended C-terminal is responsible for Plk1 binding. Cenexin1 assembles ninein and causes ciliogenesis in early stages of the cell cycle in a Plk1-independent manner. Alternatively, in the late stages of the cell cycle, G2/M phase, Cenexin1 binds to Plk1 and results in proper mitotic progression. In this study, to identify the in vivo function of Plk1 binding to phosphorylated Cenexin1 S796 residue, and to understand the in vivo functional differences between ODF2 and Cenexin1, we generated ODF2/Cenexin1 S796A/Cenexin1 WT expressing transgenic mice in a RO072 ES cell derived $ODF2^{+/-}$ knock out background. We observed a severe defect of sperm tail development by ectopic expression of Cenexin1 S796A mutant and no phenotypic differences between the ectopic expression of ODF2/Cenexin1 WT in $ODF2^{+/-}$ background and in normal wild type mice.