• Title/Summary/Keyword: Somatic embryos

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Control of MPF Activity of Recipient Oocytes and Subsequent Development and DNA Methylation of Somatic Cell Nuclear Transfer Bovine Embryos

  • Park, Joo-Hee;Choi, Yong-Lak;Kwon, Dae-Jin;Hwang, In-Sun;Park, Choon-Keun;Yang, Boo-Keun;Cheong, Hee-Tae
    • Reproductive and Developmental Biology
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    • v.33 no.4
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    • pp.223-228
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    • 2009
  • We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum-starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or $150\;{\mu}M$ roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine-treated groups (27.6%) than that of the roscovitine-treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine-treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine-treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine-treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine-treatment creates a more suitable condition for nuclear reprogramming after SCNT.

Effects of in vitro Culture Period of Reconstructed Embryos and Genetic Background of Feeder Cells on Establishment of Embryonic Stem Cells Derived from Somatic Cell Nuclear Transfer Blastocysts in Pigs

  • Han, Na Rae;Baek, Song;Lee, Yongjin;Lee, Joohyeong;Yun, Jung Im;Lee, Eunsong;Lee, Seung Tae
    • Journal of Animal Reproduction and Biotechnology
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    • v.35 no.1
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    • pp.86-93
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    • 2020
  • The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

Effects of Endoplasmic Reticulum Stress Inhibitor Treatment during the Micromanipulation of Somatic Cell Nuclear Transfer in Porcine Oocytes

  • Park, Yeo-Reum;Park, Hye-Bin;Kim, Mi-Jeong;Jung, Bae-Dong;Lee, Seunghyung;Park, Choon-Keun;Cheong, Hee-Tae
    • Development and Reproduction
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    • v.23 no.1
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    • pp.43-54
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    • 2019
  • We examined the effects of endoplasmic reticulum (ER) stress inhibitor treatment during the micromanipulation of porcine somatic cell nuclear transfer (SCNT) on the in vitro development of SCNT embryos. ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxycholic acid (TUDCA; $100{\mu}M$) were added to the micromanipulation medium and holding medium. The expression of X-box binding protein 1 (Xbp1), ER-stress-associated genes, and apoptotic genes in SCNT embryos was confirmed at the one-cell and blastocyst stages. Levels of Xbp1 splicing and expression of ER-stress-associated genes in SCNT embryos at the one-cell stage decreased significantly with TUDCA treatment (p<0.05). The expression of ER-stress-associated genes also decreased slightly with the addition of both salubrinal and TUDCA (Sal+TUD). The expression levels of caspase-3 and Bcl2-associated X protein (Bax) mRNA were also significantly lower in the TUDCA and Sal+TUD treatments (p<0.05). At the blastocyst stage, there were no differences in levels of Xbp1 splicing, and transcription of ER-stress-associated genes and apoptosis genes between control and treatment groups. However, the blastocyst formation rate (20.2%) and mean blastocyst cell number ($63.0{\pm}7.2$) were significantly higher (p<0.05) for embryos in the TUDCA treatment compared with those for control (12.6% and $41.7{\pm}3.1$, respectively). These results indicate that the addition of ER-stress inhibitors, especially TUDCA, during micromanipulation can inhibit cellular damage and enhance in vitro development of SCNT embryos by reducing stress levels in the ER.

Correlation of Oct-4 and FGF-4 Gene Expression on Peri-Implantation Bovine Embryos Reconstructed with Various Somatic Cells

  • Yoon, Byung-Sun;Song, Sang-Jin;Do, Jeong-Tae;Hong, Seung-Bum;Lee, Hoon-Taek;Chung, Kil-Saeng
    • Proceedings of the KSAR Conference
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    • 2002.06a
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    • pp.66-66
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    • 2002
  • The efficiency of animal production using cloning technology is relatively low. It is considered that the nuclear transferred (NT) embryos proceed inappropriate reconstruction with donor-recipient cell, which lead to a abnormal embryo development, and differential expression of mRNA transcript. Especially, the expression of mRNA on peri-implantation stage embryos is very important factor to decide success of implantation and ongoing pregnancy. (omitted)

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Study on Nucleo-Cytoplasmic Interaction by Somatic Cell Nuclear Transfer in Bovine (소 체내포 핵이식에 의한 핵-세포질 상호작용에 관한 연구)

  • 정희태;최종엽;박춘근;김정익;민동미
    • Journal of Embryo Transfer
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    • v.15 no.1
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    • pp.23-31
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    • 2000
  • This study was conducted to investigate the effects of quiescent treatment of donor cells and activation treatment time of recipient cytoplasm on nuclear remodeling and in vitro development of somatic cell-cloned bovine embryos. Serum starved, confluent and nonquiescent cycling adult skin cells were teansferred into enucleated oocytes. Nuclear transfer oocytes were activated at 30 min, 1 and 2 hrs after electrofusion. Some nuclear transfer embryos(23% to 35%) extruded a polar body, which was not affected by quiescent treatment of donor cells and activiation time of recipient cytoplasm. About 68% of nuclear transfer embryos fused with a serum starved cells has a chromatin clump, but which was not different from embryos fused with confluent(51%) and nonquiescent(47%) cells. The proportion of embryos with a single chromatin clump was sightly increased when nuclear transfer embryos were activated within 30 min after fusion(69%) compared to those were activated at 1 and 2 hrs after fusion, but there was not significantly different. Development rates to the blastocyst stage were 8.6% and 15.9% when serum starved and confluent cells were transferred, which were higher than that of control group. Developmental rate to the blastocyst stage was higher in embryos were activated within 30 min after fusion (17.3%) compared to those of embryos were activated at 1 and 2 hrs after fusion (P<0.05). From the present result, it is suggested that quiescent treatment of donor cells and activation time of recipient cytoplasm can affect the in vitro development. Quiescent plasm activation within 30 min after fusion could increase the number of embryos with a normal chromation structure, which results in increased in vitro development.

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Endoreduplication Pattern of Somatic Embryos and Variants Occurrence Affected by Pre-existed Endoreduplicated Cells in Doritaenopsis (Doritaenopsis 체세포배의 내배수성 특성과 절편체의 내배수성 세포에 기인한 체세포변이의 발생)

  • Park, So-Young;Paek, Kee-Yoeup
    • Journal of Plant Biotechnology
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    • v.33 no.4
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    • pp.297-302
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    • 2006
  • In general, the proliferation of orchids via somatic embryos has been used for mass production of somatic clones because of high propagation efficiency. In spite of high propagation rate, this method often brings somaclonal variation, especially polyploid frequency. Therefor we here concentrated to investigate the relationship between endopolyploidization patterns of explants and the occurrence of tetraploid variant in clonally proliferated Doritaenopsis via somatic embryo regeneration system. In the fully developed somatic embryo, upper part contained 2C to 16C while middle and lower parts showed 2C to 32C DNA content. Two-week-old embryo contained 2C to 16C, whereas those regenerated after 4 to 10-week-old contained 2C to 64C nuclei. Results showed that endoreduplication was variable depending upon tissue types, ages, and parts in one species. lower part of somatic embryo having high endoreduplication degree increased the regeneration of tetraploid variants by about 3-fold comparing to upper part of somatic embryo culture. polyploid frequency occurrence might be closely related to the high levels of endoreduplication of somatic embryos used as explant. It suggested that the upper part of somatic embryo having comparatively low endoreduplication degree is suitable for the stable in vitro propagation system.

Improvement of Black Locust(Robinia pseudoacacia L.) Through Tissue Culture. I. Micropropagation and Somatic Embryogenesis (조직배양에 의한 아까시나무(Robinia pseudoacacia L.)의 개량 I. 대량증식과 체세포배 발생)

  • Woo, Jong Ho;Choi, Myung Suk;Joung, Eun Yi;Chung, Won Il;Jo, Jin Ki;Park, Young Goo
    • Journal of Korean Society of Forest Science
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    • v.84 no.1
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    • pp.41-47
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    • 1995
  • A micropropagation system for black Locust(Robinia pseudoacacia) was established by using shoots and pin-punctured leaves of in vitro germinated seedlings. The greatest number of shoots (an average of 10.5 shoots) was obtained when shoot tips were cultured on MS medium supplemented with 1.0 mg/l BAP and 0.01 mg/l NAA. When pin-punctured leaf explants were cultured on the same medium, mean number of 13.5 shoots were produced. Shoot growth was accelerated by adding 50 mg/l of silver nitrate ($AgNO_3$), an anti-ethylene compound to the culture medium. Each shoot was excised from the mass and transferred onto half strength MS medium for rooting. Zygotic embryos at different developmental stages were cultured on LS medium supplemented with various growth regulators to induce somatic embryos. When cultured on LS medium with 1.0 mg/l 2,4-D. 14.3% of the zygotic embryos induced somatic embryos. Upon transfer onto the basal medium, somatic embryos sporadically converted into plantlets.

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Plant Production from Desiccated Somatic Embryos of Acanthopanax chiisanensis (지리오가피 (Acanthopanax chiisanensis) 체세포배의 건조처리를 통한 식물체 증식)

  • Lee, Kang-Seop;Bang, Keuk-Soo;Choi, Yong-Eui;Ahn, Byung-Yong
    • Journal of Plant Biotechnology
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    • v.30 no.4
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    • pp.381-385
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    • 2003
  • An efficient method of plant regeneration from Acanthopanax chiisanensis somatic embryos was developed. Cotyledonary somatic embryos were obtained in liquid Murashige and Skoog (MS) medium from embryogenic cell suspension cultures. They were desiccated for 0 to 72 hr and then cultured on MS medium containing NAA, BA, GA$_3$, (0-0.5mg/L). The highest multiple shoots formation (100%) was obtained from 72 hr desiccated somatic embryos on ifs medium with 0.5mg/L NAA+0.5mg/L BA or 0.5 mg/L NAA+0.5mg/L BA+0.5mg/L GA$_3$ after 6 weeks culture. Plant conversion from multiple shoots was not high. The highest plant conversion from multiple shoots was obtained on 1/3MS medium with 1.0mg/L GA$_3$. Converted plantlets were transferred to ex vitro condition and the highest survival rate (70%) of the plantlets was obtained on plastic pots containing vermiculite and sand. These results indicate that micropropagation procedure can be applied for an efficient mass propagation of Acanthopanax chiisanensis.

A Study on Culture Environments of In Vitro Matured/In Vitro Fertilized Bovine Embryos I. Influence of Somatic Cells, Growth Factors or Culture Media on In Vitro Maturation of Bovine Oocytes (소 체외수정란의 발생배양에 적합한 배양환경 조성 연구 I. 체세포, 성장인자 또는 배양액 종류가 난포란의 체외성숙에 미치는 효과)

  • 이명식;박수봉;박진기;장원경;민관식;백광수;성환후;박용윤
    • Korean Journal of Animal Reproduction
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    • v.22 no.1
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    • pp.95-99
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    • 1998
  • Three experiments were conducted with follicular oocytes, to compare some somatic cells, growth factors and media for in vitro maturation of bovine oocytes. In the first experiment, the type of somatic cells had no effects on in vitro maturation of bovine follicular ooctyes. In the second experiment, oocytes were matured in TCM199 su, pp.emented with growth factors on IVM of bovine follicular oocytes, then all were co-cultured with cumulus cells. The proportion of used oocytes that developed to expanding blastocysts was 22.2%, 20.2%, 17.7%, 22.2%, 24.4% and 20.2% after maturation in TCM199 su, pp.emented with control, insulin, IGF-I, IGF-Ⅱ, FGF and EGF, respectively. In the third experiment, oocytes were matured in BO, Ham's F10 and TCM199, then all were fertilized in BO, and embryos cultured in BO, Ham's F10 and TCM199, respectively. Cleavage rates in BO were 90%, had higher than in Ham's F10(80%) or in TCM199(64%). But production of expanding blastocysts in TCM199(21%) or Ham's F10(20.6%), had higher than in BO(4.6%).

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Aberrant Distributions of ICM Cells in Bovine Blastocysts Produced by Somatic Cell Nuclear Transfer

  • D. B. Koo;Y. K. Kang;Park, Y. H.;Park, J. S.;Kim, H. N.;D. S. Son;Y. M. Han;Lee, K. K.
    • Proceedings of the KSAR Conference
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    • 2001.03a
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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.

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