• Molecules and Cells
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• 제40권2호
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• pp.117-122
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• 2017

Despite the fact that porcine embryonic stem cells (ESCs) are a practical study tool, in vitro long-term maintenance of these cells is difficult in a two-dimensional (2D) microenvironment using cellular niche or extracellular matrix proteins. However, a three-dimensional (3D) microenvironment, similar to that enclosing the inner cell mass of the blastocyst, may improve in vitro maintenance of self-renewal. Accordingly, as a first step toward constructing a 3D microenvironment optimized to maintain porcine ESC self-renewal, we investigated different culture dimensions for porcine ICM-derived cells to enhance the maintenance of self-renewal. Porcine ICM-derived cells were cultured in agarose-based 3D hydrogel with self-renewal-friendly mechanics and in 2D culture plates with or without feeder cells. Subsequently, the effects of the 3D microenvironment on maintenance of self-renewal were identified by analyzing colony formation and morphology, alkaline phosphatase (AP) activity, and transcriptional and translational regulation of self-renewal-related genes. The 3D microenvironment using a 1.5% (w/v) agarose-based 3D hydrogel resulted in significantly more colonies with stereoscopic morphology, significantly improved AP activity, and increased protein expression of self-renewal-related genes compared to those in the 2D microenvironment. These results demonstrate that self-renewal of porcine ICM-derived cells can be maintained more effectively in a 3D microenvironment than in a 2D microenvironment. These results will help develop novel culture systems for ICM-derived cells derived from diverse species, which will contribute to stimulating basic and applicable studies related to ESCs.

• Reproductive and Developmental Biology
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• 제29권1호
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• pp.37-41
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• 2005

본 연구는 한우 수정란의 체외생산에 있어서 효율과 품질의 향상을 위해서, 미성숙 난포란을 회수하는 난소의 형태와 배양 용기로서 straw의 효과를 검토하였다. 난소의 형태에 따른 수정율은 전 군에서 70.3∼84.1%로서 비슷한 경향이었다. 8세포기 및 배반포기 발달율은 황체와 난포가 모두 존재하지 않는 대조군이 가장 높았다. 배반포의 inner cell mass(ICM), trophectoderm(TE), total cell number(TCN) 및 ICM/TCN 비율은 낭종군과 퇴행황체군이 다른군에 비하여 높은 경향이었다. 체외성숙에 이용하는 배양용기에 따른 수정율은 0.5 ㎖ straw 군이, 8세포기 발달율은 대조군이 가장 높았으나, 배반포기 발달율은 23.1∼30.7％로서 각 군 간에 비슷한 경향이었다. 한편 각각의 배양 용기에서 유래된 배반포의 ICM, TE, TCN 및 ICM/TCN 비율은 유사한 경향이었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.20-20
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• 2001

It has been reported that cloning cattle is inefficient. One of the problems was placental abnormality, finally resulting in fetal mortality after transfer of nuclear transfer (NT) bovine embryos. This study was focused on the allocations of embryonic cells to the inner cell mass (ICM) or to the trophectoderm(TE) in NT bovine blastocysts. Somatic cells were derived from a Day 45 fetus of gestation, individually transferred into enucleated oocytes and developed to the blastocyst stage in vitro. Differential staining was used to assess the qualify of blastocysts derived from NT, IVF and in vivo. Development rate of NT embryos to blastocysts (25.0%, 41/164) was similar to that of IVF embryos (28.7%, 49/171). The total cell number of NT blastocysts (101.3$\pm$45.9) was not different compared with that of IVF embryos (107.9$\pm$34.2, P>0.05), but was lower than in vivo embryos (122.5$\pm$21.6, P<0.05). Ratio of ICM/total cells was higher in NT embryos (51.6$\pm$ 18.6%) than in IVF and in vivo embryos (42.3$\pm$ 15.3% and 34.9$\pm$8.9%, respectively) (P<0.05). Most IVF (56.8%, 25/44) and in vivo blastocysts(80.8%, 21/26) was distributed in the proportion of ICM/total cells ranging from 20 to 40% group. However, most NT blastocysts was biased in the 40-60%(34.1%, 15/44) and >60% (31.8%, 14/44) groups. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocation of NT embryos to the ICM cells.

• 한국수정란이식학회지
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• 제31권1호
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• pp.27-32
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• 2016

This study was conducted to evaluate the effects of different volume ($100{\mu}l$ vs. 2 ml) of microdrop culture on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. Blastulation rate was not different between groups ($58.4{\pm}2.9%$ vs. $61.2{\pm}4.8%$). Zona hatched rate ($38{\pm}15.4%$ vs. $27{\pm}3.4%$) and attached rate ($55{\pm}13.9%$ vs. $46{\pm}3.9%$) did not differ by the volume of culture media. Total cell numbers ($59.8{\pm}9.7$ vs. $70.3{\pm}8.7$), ICM cell numbers ($15.8{\pm}0.6$ vs. $16.8{\pm}1.5$), TE cell numbers ($44.0{\pm}9.7$ vs. $53.6{\pm}7.3$), % ICM ($26.4{\pm}2.9%$ vs. $23.8{\pm}3.3%$) and ICM:TE ratio ($1:2.8{\pm}0.4$ vs. $1:3.2{\pm}0.6$) were not different between groups (i.e., $100{\mu}l$ vs. 2 ml). These results show that the capacity of the culture medium did not effect the cell numbers of B6D2F1 mice blastocysts. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• 한국해안해양공학회지
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• 제18권1호
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• pp.43-52
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• 2006

해수소통을 전제한 경우 새만금호 내측의 염분변화가 만경강의 평수량 및 홍수량 유입에 따라 혼합 확산되는 것을 평가하고, 내부 개발이후 배수수문을 통한 새만금호소수 방류시 관리수위별로 저염수가 외해에 미치는 영향범위를 평가하기 위한 수치모형실험이 실시되었다. 해석에는 2차원 ADCIRC 및 3차원 TIDE3D모형과 3개 연직층을 고려한 3차원 CE-QUAL-ICM모델이 이용되었다. 내부의 확산 모의결과는 시간이 지남에 따라 외해에서 유입되는 염수가 점차 혼합확산되며, 최소 1개월 이상 경과되어야 만경호측에 외해수가 혼합되고, 5개월 지속되어야만 동진호측까지 혼합된다. 호내측의 수위를 관리하기 위해 신시 또는 가력수문을 개방하면 유속이 지수적으로 감소되나 갑문으로부터 전면 10km 이상까지 0.5m/s의 유동장이 형성된다. 이러한 결과는 저염수 방류로 인한 영향이 주기적으로 낙조시에 발생되어 새만금 방조제 전면의 해수순환과 유동 및 생태환경에 적지 않은 영향을 미칠 개연성을 제시하는 것으로 해석된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.68-68
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• 2001

Blastocyst formation, consisting of the inner cell mass (ICM) and trophectoderm (TE), is the first differentiation process during embryonic development in mammals. It has been hypothesized that the proportion of ICM to TE in the blastocyst may be crucial for subsequent developmental competence of early embryos, which it may be expressed as a sensitive indicator for evaluating in vitro systems. In this study ICM/total cell ratio of nuclear transfer (NT) embryos was compared with IVF-derived and in vivo embryos. Somatic cell nuclei obtained from a fetus at Day 40 of gestation were transferred into the enucleated oocyte and then cultured in NCSU 23 medium for 6 days as previously described (Koo et al., Biol. Reprod. 2000; 63:986-992). ICM and TE cells of blastocysts were determined by using a differential staining method (Han et al., Biol. Reprod. 1999; 60:1110-1113). Development rate (9.8$\pm$2.5%, 23/225) to the blastocyst stage of NT embryos was lower than IVF embryos (23.8$\pm$2.7%, 53/223). Thus, a difference was detected in the in vitro developmental rate to blastocyst stage between NT and IVF-derived embryos (P<0.05). In the next experiment, we investigated ICM and TE nuclei to assess the quality of blastocysts that produced by NT, IVF and in vivo, respectively. NT blastocysts (27.6$\pm$8.3) showed a smaller total cell number than IVF-derived (42.6$\pm$17.4) and in vivo embryos (283.9$\pm$103.5) (P<0.05). Ratios of ICM/total cells in NT, IVF and in vivo blastocysts were 15.1$\pm$ 18.6% (n=56), 12.3$\pm$9.2% (n=57) and 30.4$\pm$6.8% (n=40), respectively. Individual blastocysts for the ratio of ICM/total cells were assigned to 3 groups (I; <20%, II; 20 to 40% and III;>40%). As the results, most in vivo blastocysts (97.5%, 39/40) were distributed into group II while most NT (78.6%, 44/56) and IVF-derived blastocysts (82.5%, 47/57) were allocated to group I. Thus, our data show that NT or IVF-derived embryos have aberrant morphology during early development in vitro systems, suggesting that these anomalies may result in developmental failures of the NT embryos to term.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.66-66
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• 2003

The aim of this study was to investigate effect of different energy substrates on embryonic development of mouse embryos. Two cell embryos, recovered from ICR female mice (4 weeks old) at 44~52hrs after hCG injection (mated just after hCG injection), were cultured fur 72 hrs in the medium (MEM) supplemented with the three different energy substrates [glucose(G), pyruvate(P) and lactate(L)] and combinations (Control: 0 mM: group A: G 0.5; B: G 3.15; C: P 0.1; D: P 0.32; E: L 5.87; F: L 10.5; G: G0.5+P0.32+L10.5; H: G3.15+P0.1+L5.87; I: G0.5+P0.1+L5.87; J: G3.15+P0.32+L10.5). Blastocysts were stained differentially using PI and bisbenzimide. The 69.8% of the 2 cell embryos cultured in group F were developed the blastocysts. This was the highest (NS) than all other tested groups (44.2~62.8%). Blastocysts, cultured in the group E (60.4$\pm$26.9) and G (58.1$\pm$26.3), had significantly(p<0.05: group E vs. control, B, C, D; G vs. control, A, B, C, D) higher mean cell number compared with the other (42.6$\pm$25.8 ~ 55.2$\pm$31.3) and control (42.6$\pm$25.8) was at the basal level. The proportion of ICM (% ICM of total cells) in blastocysts cultured in group B (26.0$\pm$9.5%), C (29.6$\pm$22.8%) and J (26.0$\pm$11.8%) were significantly higher (p<0.05: control vs. group B, C, J: A vs. C, J; C vs. D, E, I) than those of other tested groups (15.0$\pm$10.6 ~ 23.8$\pm$ 12.9％) and control (15.0$\pm$10.6％) was at the basal level. These results showed that energy substrates supported the development of mouse 2 cell embryos, especially with greater embryo development in high dose of lactate added to media.

• 천문학회보
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• 제45권1호
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• pp.55.2-55.2
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• 2020

Radio relics in the outskirts of galaxy clusters are interpreted as synchrotron radiation due to the relativistic electrons produced via diffusive shock acceleration (DSA) in shocks with low sonic Mach numbers, Ms ≤ 3 in high beta ICM plasma. Electron injection into the DSA process at such weak shocks is one of the key elements, which has yet to be fully understood. In this study, we explore the nature of kinetic microinstabilities excited in weak quasi-perpendicular shocks through 2D particle-in-cell simulations. We find Alfven-ion cyclotron (AIC), whistler, and mirror instabilities can be triggered by ion and electron temperature anisotropy in the immediate downstream of supercritical shocks with Ms > Mcrit ~ 2.3. In particular, AIC instability causes rippling of the shock surface, which in turn generates plasma waves on multi-scales and faciliates the electron preacceleration. Our results may contribute to understanding the origins of radio relics.

• 한국수정란이식학회지
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• 제31권1호
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• pp.19-25
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• 2016

This study was conducted to evaluate the effects of type of culture media (BM, G2, OS, TCM, and MEM) on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. In vitro maturation was highest in BM followed by the order of OS, MEM, TCM and G2 ($90{\pm}2.8%>88{\pm}3.2%>85{\pm}4.9%>78{\pm}10.2%>64{\pm}7.7%$, respectively). To note, the G2 group was statistically different compared to other groups (p<0.05). On the other hand the fertilization rate was highest in the G2 group followed by BM, OS, TCM, and MEM ($87{\pm}7.2%>85{\pm}6.9%>74{\pm}14.0%>71{\pm}13.8%>2{\pm}1.4%$, respectively). The MEM group was significantly lower compared to other groups (p<0.05). The developmental rate was highest in the OS group followed by the G2 group and the BM group albeit no statistical significance was noted ($73{\pm}11.6%>71{\pm}9.2%>66{\pm}10.4%$). Of note, all cells of the TCM and MEM groups were died during embryonic development. The zona hatched rate ($51{\pm}9.8%$ vs. $50{\pm}9.1%$ vs. $47{\pm}7.2%$ for BM, G2, and OS respectively) and attached rate ($45{\pm}12.3%$ vs. $38{\pm}16.1%$ vs. $37{\pm}11.5%$ for BM, G2, and OS respectively) were not different amongst groups. No difference was found in total cell numbers ($74{\pm}13.9$ vs. $64{\pm}9.2$ vs. $76{\pm}6.7$ for BM, G2, and OS respectively), ICM cell numbers ($20{\pm}1.9$ vs. $14{\pm}1.8$ vs. $15{\pm}2.1$), TE cell numbers ($55{\pm}12.5$ vs. $49{\pm}10.7$ vs. $61{\pm}5.9$), % ICM ($30{\pm}2.8%$ vs. $24{\pm}7.0%$ vs. $22.8{\pm}2.2%$) and ICM:TE ratio ($1:2{\pm}0.5$ vs. $1:3.1{\pm}0.8$ vs. $1:3.1{\pm}0.5$) amongst groups. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• 한국수정란이식학회지
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• 제33권4호
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• pp.205-210
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• 2018

This study was performed to investigate the effect of types of oil (OVOIL vs. OIL) on B6D2F1 mice oogenesis. In this study, B6D2F1 F1 mice were used in order to maximize oogenesis. The expansion rate of cumulus cells ($82.0%{\pm}0.2$ vs. $78.0%{\pm}0.1$), in vitro fertilization rate ($92.0%{\pm}0.1$ vs. $88.0%{\pm}0.1$), developmental rate ($91.0%{\pm}0.1$ vs. $87.0%{\pm}0.2$), blastocysts formation rate ($56.0%{\pm}0.1$ vs. $57.0%{\pm}0.1$), and zona hatched rate($41.4%{\pm}0.2$ vs. $24.0%{\pm}0.1$) were not different between groups (NS; P>0.05). However, there was a significant difference in maturation rate; the OVOIL group showed higher maturation rate compared to that of the OIL group ($96.0%{\pm}0.1$ vs. $87.0%{\pm}0.1$; P<0.05). In the blastocysts cell numbers, the total cell numbers ($83.9{\pm}26.1$ vs. $56.9{\pm}23.9$), ICM cell numbers ($15.7{\pm}8.8$ vs. $6.3{\pm}3.5$), TE cell numbers ($68.3{\pm}25.7$ vs. $50.7{\pm}24.1$), % ICM ($21.6%{\pm}0.1$ vs. $12.7%{\pm}0.1$), and the ratio of ICM:TE ($1:6.2{\pm}6.5$ vs. $1:10.3{\pm}7.0$) were significantly higher in the OVOIL group than the OIL group (P<0.05). These results suggested that it is expected to achieve the more developmental ability of B6D2F1 mice depending on the type of oil (OVOIL vs. OIL). In addition, the results can provide essential information for culture condition on B6D2F1 mice. Henceforth, thus, it is expected that these results herein might be used for in vitro culture of human embryos.