• 한국가축번식학회지
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• 제21권2호
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• pp.85-93
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• 1997

These experiments were carried out to examine the development capacity of sexed and then bisected embryo from 8-cell to morula stage. Antisera to histocompatibility-Y(H-Y) antigen were prepared in inbred SD female rat by repeated immunization of spleen cell or testis supernatant from males of same strain. Male and female embryos were separated by delaying development of embryos against H-Y antibody. After sexing, rabbit embryos were bisected and aggregated. The results obtained from the these experiments were summuerized as follows: 1. When mouse and rabbit 8-, 16-cell and morular embryos were culature in H-Y antiserum, the ratio of embryo which has developed to hatching blastocyst was 53.4, 46.3 and 57.4% in mouse embryos, and 49.0, 52.0 and 61.0% in rabbit embryo, respectively. The ratio of mouse and rabbit embryos developed to hatching blastocyst showed nearly natural sex rate(50%), except rabbit mourla showed a little higher ratio(61.0%) as compared to natural sex ratio. 2. When rabbit demi-embryos from 8-, 16-cell embryo and morula were cultured, the percentage of demi-embryos was 70, 68 and 58% without zona pellucida removed, and 62, 69 and 51% with zona pellucida. The rate of aggregation was higher in 8- and 16-cell demi-embryos than in morula demi-embryo. 4. When sexed-demi-embryo was aggregated with another demi-embryo with demi-embryo with same sex, the rate of embryo developed to blastocyst was 60, 50 and 25%, respectively. Eight-cell demi-embryo showed highest rate. In conclusion, it showed that H-Y antiserum which was made by rat spleen cell enabled sexing rabbit embryos. And when rabbit sexed 8-, 16-cell and morula demi-embryo were aggregated, they were developed to eu-blastocyst which suggested the potential of sexed embryo manipulation.

• 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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• 제9권1호
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• pp.111-116
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• 1994

This study was carried out to investigate the aggregation rate of isolated mouse 2-, 4- and 8-cell stage blastomeres in phytohemagglutinin(PHA) solution. The morphologically normal embryos were collected from the oviduct of superovulated female mouse by flushing with M2 and the zona pellucida of embryos were removed with 0.5% pronase. The blastomeres were isolated by pipetting after plunging into Ca++-Mg++free PBS for 20 min. The result showed that aggregation rate in 0.5% (84.9~93.1%) was higher than that in 1.0% PHA(76.0~82.1%). Optimal aggregation time was 60min (83.9~100.0%) when compared with 30min (78.8~87.5%). Developmental to blastocyst in recombinated blastomeres was higher under conditions of 0.5% PHA solution and 60-min aggregation than that under other conditions.

• 대한수의학회지
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• 제63권1호
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• pp.3.1-3.9
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• 2023

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode's solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%-38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 ㎛ vs. 230-234 ㎛). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%-6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

• 한국가축번식학회지
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• 제24권4호
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• pp.451-455
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• 2000

본 연구는 TT2 embryonic stem(ES) cell을 이용하여 chimeric mouse를 생산하는데 있어서 더욱 간편한 공배양방법 개발하기 위하여 수행되었다. 유전자 적중 생쥐의 개발은 유전자의 기능을 연구하는데 매우 중요한 수단으로 이용되고 있다. 이러한 생쥐의 개발에 있어서 chimeric mouse를 생산하는 과정은 ES cell의 종류의 차이는 있지만 주로 배반포기의 수정란에 ES cell을 주입하고 있다. 이 기술은 고가의 미세조작장치 뿐만 아니라 고도의 기술을 요하고 있다. 그러므로 본 연구에서는 TT2 ES cell를 8세포기 수정란과 공배양할 때의 필요로 하는 적절한 ES cell의 수를 검증함으로써 chimeric mouse의 생산 효율을 높일 수 있었다. 각각 0.5$\times$$10^{6}$, 1$\times$$10^{6}$과 2$\times$$10^{6}$$m\ell$의 ES cell을 8 세포기의 수정란과 공배양하였을때 0.5$\times$$10^{6}$과 1$\times$$10^{6}$$m\ell$에서 높은 배반포기로의 발달율을 나타내었다. 또한 가임신된 생쥐에 이들 배반포기를 이식한 결과 1$\times$$10^{6}$$m\ell$에서 높은 chimeric mouse 생산 효율을 나타내었다. 이러한 결과는 적절한 수의 ES cell과 수정란을 공배양함으로써 매우 간단하게 효율 좋은 chimeric mouse을 얻을 수 있음을 제시하고 있다.

• 한국동물학회지
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• 제33권3호
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• pp.310-321
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• 1990

연골세포 분화기구 연구의 기초단계로서 미세 세포배양법을 정립하였으며, 세포의 응집정도와 연골분화의 관계를 조사하기 위하여 계배 limb bod 간충직세포를 여러가지 농도로 micromass 배양하면서 세포농도에 따른 세포증식정도와 proteoglycan에 결합된 alcian blue의 양 및 [35 S] sulfate의 sulfate proteohlycan에 표지되는 속도를 측정하고, 면역조직화학법을 이용하여 type II collegen의 발현을 관찰하였다. 각 농도별로 배양한 Hambruger-Hamilton stage 23/24 간충직세포 중 5. $\times$ 106 cells/ml 이상의 농도로 배양한 세포는 연골세포로 분화하였으나, 저농도로 배양한 세포는 분화하지 않았다. 반면에 stage 18/19 간충직세포는 분화단계 중에서 동일세포들끼리 응집되는 단계이며 stage 23/24 간충직세포 이 단계를 지나 분화능을 갖는 세포응축의 단계인 것으로 생각된다. 본 연구 결과 세포응집 및 분화능은 간충직세포의 발생시기에 따라 다르며, 분화능을 갖기 위해서는 세포응집이 선행조건이고, 그 적정 미세 배양 농도는 5-10 $\times$ 106 cells/ml임을 알았다. 한편, hyaluronidase의 활성은 stage 23/24세포 배양 전 과정에서 비교적 일정한 것으로 나타나 이 시기의 세포분화에는 별로 중요하지 않는 것으로 보인다.

• Molecules and Cells
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• 제38권12호
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• pp.1096-1104
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• 2015

Particulate $matter_{2.5}$ ($PM_{2.5}$) is notorious for its strong toxic effects on the cardiovascular, skin, nervous, and reproduction systems. However, the molecular mechanism by which $PM_{2.5}$ aggravates disease progression is poorly understood, especially in a water-soluble state. In the current study, we investigated the putative physiological effects of aqueous $PM_{2.5}$ solution on lipoprotein metabolism. Collected $PM_{2.5}$ from Seoul, Korea was dissolved in water, and the water extract (final 3 and 30 ppm) was treated to human serum lipoproteins, macrophages, and dermal cells. $PM_{2.5}$ extract resulted in degradation and aggregation of high-density lipoprotein (HDL) as well as low-density lipoprotein (LDL); apoA-I in HDL aggregated and apo-B in LDL disappeared. $PM_{2.5}$ treatment (final 30 ppm) also induced cellular uptake of oxidized LDL (oxLDL) into macrophages, especially in the presence of fructose (final 50 mM). Uptake of oxLDL along with production of reactive oxygen species was accelerated by $PM_{2.5}$ solution in a dose-dependent manner. Further, $PM_{2.5}$ solution caused cellular senescence in human dermal fibroblast cells. Microinjection of $PM_{2.5}$ solution into zebrafish embryos induced severe mortality accompanied by impairment of skeletal development. In conclusion, water extract of $PM_{2.5}$ induced oxidative stress as a precursor to cardiovascular toxicity, skin cell senescence, and embryonic toxicity via aggregation and proteolytic degradation of serum lipoproteins.

• 한국미생물·생명공학회지
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• 제46권4호
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• pp.372-376
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• 2018

연(Nelumbo nucifera Gaertner)으로부터 신규의 유용 생리활성을 확인하기 위해, 연잎, 연자방, 연자육, 연자심, 우절 및 연근으로부터 각각 ethanol 추출물을 제조하고, 현재까지 보고되지 않은 항혈전 활성을 평가하였다. 그 결과, 연근, 연잎 및 연자심 추출물은 항응고 활성이 인정되지 않았으나, 우절, 연자방 및 연자육 추출물에서는 강력한 TT, PT, aPTT 연장효과를 나타내었으며, aspirin 보다 강력한 항응고 활성을 나타내었다. 또한 혈소판 응집저해 활성 평가 결과, 우절, 연잎, 연자방, 연자육 추출물에서 응집저해가 나타났으며, 가장 강력한 응집저해는 연자방과 연자육에서 확인된 바, 이는 아스피린에 필적하는 강력한 활성이었다. 상기 활성 추출물은 1.0 mg/ml 농도까지 인간 적혈구 용혈활성이 없음을 확인하여, 우절, 연자방 및 연자육 추출물이 신규의 항혈전제로 사용 가능함을 제시하였다.

• 대한수의학회지
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• 제31권4호
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• pp.523-527
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• 1991

The effects of ethylene glycol, DMSO and glycerol as cryoprotectants and the effect of concentrations(0, 0.25, 0.5 and 1.0M) of sucrose in the diluent on the viability of the aggregated morulae frozen rapidly in liquid nitrogen$(LN_2)$ vapour were examined. The morulae were produced by aggregation of ICR and CBA mice embryos at 8-cell stage. Before freezing the embryos were equilibrated in 1.5M cryoprotectants+0.25M sucrose in oae-step or in 3.0M cryoprotectants+0.25M sucrose in two-steps. The embryos were pipetted into the freezing medium fraction of 0.25ml plastic straws. The straws were frozeu by directly transfer into $LN_2$ vapour(about lcm above $LN_2$) for 2 minutes, and then plunged into $LN_2$. After thawing the cryoprotectants were diluted with 0, 0.25, 0.5 or 1.0M sucrose solution. The post-thawed in vitro viability of the aggregated embryos was significantly dependent on the type and concentration of cryoprotectants in the freezing medium and also on the concentration of sucrose in the diluent. When the aggregated embryos were equilibrated in 1.5M cryoprotectants +0.25M sucrose in one-step and diluted with 0.5M sucrose after thawing, the survival rate of the embryo5 was significantly(p<0.05) higher in DMSO(62.5%) or ethylene glycol(52.2%) than in glycerol(33.3 %). In the case that the concentration of the cryoprotectants was raised to 3.0M in two-steps, thc higher survival rate of the embryos was obtained in ethylene glycol or glycerol than in DMSO followed by diluting them with 0.5 or 1.0M sucrose after thawing(p<0.01).

• Asian-Australasian Journal of Animal Sciences
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• 제14권4호
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• pp.455-461
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• 2001

The effect of exogenous gonadotrophins on superovulation in rabbits was examined. One hundred and sixteen sexually mature California, Chinchilla and New Zealand White rabbits were randomly allocated to control (100 IU hCG), PMSG-treated (100 IU HCG following 150 IU PMSG) and FSH-treated groups (0.3 mg/head /12 h for 3 days followed by 100 IU hCG). All does were mated after hCG injection and were sacrificed or laparotomized within 1 to 4 days postcoitus for counting the number of ovulation points. The number of ovulations was higher in FSH-treated animals than in the control and PMSG-treated groups (37.2 vs. 10.4 and 14.5, p<0.05). Follicle haemorrhagicum was observed in many cases in the PMSG-treated group. No significant difference in ovulation number was observed between left and right ovaries regardless of gonadotropin treatment. In another experiment, 2-cell stage embryos were collected at 26 h postmating and blastomeres were separated by mechanical pipetting or gentle pressure with a fine glass needle. Aggregated or chimeric embryos were produced from two single blastomeres from two breeds, New Zealand White and Chinchlla, with different coat colors. All the embryos were cultured in Ham's F-10 medium supplemented with 1.5% BSA (bovine serum albumin fraction V) and 10% PRS (pregnant rabbit serum), and incubated in a humidified atmosphere with 5% $CO_2$ at $38^{\circ}C$. After development to morula or early blastocyst, the embryos were transferred into the oviducts of recipient does. Results showed that 7 out of 10 does (70%) receiving intact embryos (control) became pregnant and 41 kits were delivered. However, no pregnancy was obtained from the recipient of either denuded demi- or aggregated embryos. It is suggested that embryos without zona pellucida could not develop to term in rabbits.