• Journal of Forest and Environmental Science
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• 제23권1호
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• pp.5-9
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• 2007

Zygotic embryos just after harvest of seeds were immature globular to heart stage. Maturation of zygotic embryos rapidly proceed when zygotic embryos together with small excised parts of endosperm were cultured on 1/3-strength MS solid medium with 2% sucrose, and the zygotic embryos were germinated within two months. Embryogenic callus was formed from the excised segments of germinating zygotic embryos of Eleutherococcus pedunclus on Murashige and Skoog (MS) medium with $4.5{\mu}M$ 2,4-D. The embryogenic callus formation occurred at a low frequency (less than 7%) from hypocotyl segments. The embryogenic calli were maintained on the same medium as primary medium. High frequency somatic embryogenesis was obtained after the cells were transferred to medium lacking 2,4-D. Cotyledonary embryos were germinated and converted into plantlets on medium with $20{\mu}M$ $GA_3$. Embryogenic callus and somatic embryos were produced spontaneously on the surfaces of roots and/or hypocotyls of plantlets. The frequency of embryogenic callus formation was 85% in roots and 34% in hypocotyls. Therefore maintain of cell lines performed very easily. Plantlets with developed epicotyls at more than 3 cm acclimatized at high frequency (89%). While plantlets with small epicotyls (less than 1 cm) were acclimatized at low rate (32%). The soil survived plantlets produced new sprouts after over wintering in the field.

• 한국수정란이식학회지
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• 제10권1호
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• pp.65-72
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• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• 한국가축번식학회지
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• 제20권2호
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• pp.163-170
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• 1996

The effect of several potential antioxidants and amino acids were examined as a means of increasing the development of in vitro matured and in vitro fertilized oocytes into morulae or blastocysts. 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 varing concentraton of the antioxidants and amino acid in a gas phase consisting of 5% CO2, high humidified air. At 5~6 days, embryo developments were reduced, and embryos were fixed and stained with Hochest 33342 DNA stain to facilitate counting of cells. In experiment 1, the proportion of embryos developed to morulae and blastocysts in CR1aa containing 1mM, 2.5mM taurine (22.6% and 20.4%) was slightly higher than those of 0, 5 and 10mM Taurine (5.7, 5.7 and 3.9%, P<0.05). In experiment 2, addition of glutathione did not improve blastocyst development (P>0.05). In experiment 3, concentations of superoxide dismutase(SOD) ranging from 300 to 1,000 U did not affect the propotion of embryos developing into blastocysts (P>0.05). In experiment 4, addition of 250 U catalase(38.5%) was slighty higher than those of 0, 500 and 1,000U. In experiment 5, the proportion of embryo developed beyond morula stage in CR1aa with taurine plus EDTA was slighty higher than other treatments(15.7, 26.0 and 29.2%), there were no significantly increases in cell number among treatments(P>0.05). These results are indicating that antioxidants and amino acids can increase the proportion of embryos that develop into morulae and blastocysts, but did not increas in cell number of blastocysts.

• 한국동물학회지
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• 제32권4호
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• pp.404-411
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• 1989

In this study, we attempted to determine the precise patterns of protein synthesis during the second cleavage in mouse. F1 hybrid 2-cell embryos showing a highly synchronized cell cycle and outbreed ICR strain 2-cell embryos were used. The patterns of protein synthesis during the second cleavage showed the sequential changes in the F1 hybrid and ICR strain 2-cell embryos. Moreover, we examined the effects of mitotic and transcriptional inhibitors such as colcemid and a-amanitin on the protein synthesis in the late 2-cell embryos of ICR strain. Treatment of colcemid (0.1mg/ml) blocked the second cleavage, but did not affect on the change of protein synthesis. However, treatment of a-amanitin induced the synthesis of two set of polypeptides without affecting on synthesis of other proteins and cleavage. It thus seems that the appearance of a-amanitin-sensitive proteins may be not involved in the second cleavage. Therefore, these results indicate that the second cell cycle in mouse embryos appears to be regulated at post transcriptional level, presumably independent on the expression of embryonic genome. 본 연구는 생쥐 배아의 2세포기 분열과정중 단백질 합성양상과 단백질합성에 미치는 colcemid와 $\alpha$-amanitin의 영향을 조사하였다 이를 위하여 체내 수정된 ICR strain의 2세포기 배아와 매우 일치된 초기배아 분열양상을 보여주는 체외 수정된 F1 (C57BL x CBA) hybrid 2세포기 배아를 사용하였다. 두 종류의 2세포기 배아에서 단백질 합성은 분열단계에 따라서 매우 일치된 변화를 보여 주었다. 또한 유사분열 억제제인 colcemid (0.1mg/ml)의 처리는 2세포기 배아분열을 억제하였으나, 단백질 합성에는 아무런 변화를 주지 못하였다. 그리고 후기 2세포기 배아에 전사 억제제인 a-amanitin (100mg/ml)을 처리하였을 때 세포분열이나 다른 단백질의 합성에는 아무런 영향이 없이 단지 두개의 단백질의 합성만을 유도하였다. 이는 아마도 a-amanitin의 stress효과에 기인하는 것으로 추측된다. 따라서 생쥐 2세포기 배아의 분열과정은 배아게놈의 유전자 발현과는 무관하게 이미 합성되어 존재하는 mRNA에 의하여 조절되는 것으로 사료된다.

• 한국가축번식학회지
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• 제11권3호
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• pp.161-169
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• 1987

This experiment was carried out to investigate the optimal cooling rate and the plunging temperature into liquid nitrogen of the 8-cell hamster embryos. The female hamsters were induced to superovulate by intraperitoneal injection of 30 i.u. PMSG. Embryos were flushed from oviduct and uterine horn. Embryos were frozen and incubated with a modified Dulbecco's phosphate buffered saline, and equilibrated with 1.5M-dimethyl sulfoxide by a 3-step procedure. The cooling rate of samples was 1$^{\circ}C$/min from room temperature to -5$^{\circ}C$ and the samples were seeded at -5$^{\circ}C$. The plunging temperatures into liquid nitrogen were -20, -25, -30, -35, -40, -45, -50 and -55$^{\circ}C$ at 0.3$^{\circ}C$/min, 0.5$^{\circ}C$/min and 1$^{\circ}C$/min cooling rates, respectively. This mean numbers of ovulation points and recovered embryos were 59.4 and 48.4 appearing 81.6% recovery rate. The percentage of 8-cell embryos in recovered embryos was 68.2. The survival rates of embryos plunged at -45$^{\circ}C$ were 73.5% at 0.3$^{\circ}C$/min, 44.8% at 0.5$^{\circ}C$/min and 30.3% at 1$^{\circ}C$/min cooling rates, respectively. This mean numbers of ovulation points and recovered embryos were 59.4 and 48.4 appearing 81.6% recovery rate. The percentage of 8-cell embryos in recovered embryos was 68.2. The survival rates of embryos plunged at -45$^{\circ}C$ were 73.5% at 0.3$^{\circ}C$/min, 44.8% at 0.5$^{\circ}C$/min and 30.3% at 1$^{\circ}C$/min cooling rates, respectively. The survival rates at 0.3$^{\circ}C$/min were significantly high. Under the condition of 0.3$^{\circ}C$/min cooling rate, the survival rates of embryos according to the plunging temperature were 70.0% and 73.5% at -40 and -45$^{\circ}C$, and those were higher than other plunging temperatures. Under the condition of 0.5$^{\circ}C$/min and 1$^{\circ}C$/min cooling rates, the survival rates according to the plunging temperatures were lower than the cooling rate of 0.3$^{\circ}C$/min, showing the similar tendency at all the plunging temperatures. In conclusion, 8-cell hamster embryos showed the best survival rates at 0.3$^{\circ}C$/min cooling rate and -45$^{\circ}C$ plunging temperature.

• Clinical and Experimental Reproductive Medicine
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• 제27권4호
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• pp.381-385
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• 2000

Objective: To verify the effect of two forms (growth factor and growthfactor-reduced) of solubilized Matrigel on the development in mouse preimplantation embryos. Methods: Late 2-cell stage eggs were cultured through the blastocyst stage in the presence of GF- or GFR-Matrigel (0.5%, v/v). Morphological development, cell number and % apoptotic nuclei of blastocyst were measured by Roecst staining and TUNEL of nuclei. Results: Morphological development, number of cells per embryo was significantly increased in the presence of GF- or GFR-Matrigel. Culture of the embryos in the GF-Matrigel gave the best result. Conclusion: Low concentration of solubilized Matrigel improved development of mouse embryos regardless of growth factor content of the Matrigel. Growth factors and extracellular matrix protein included in the Matrigel synergistically potentiated the development of mouse embryos.

• 한국가축번식학회지
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• 제19권2호
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• pp.81-88
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• 1995

Culture medium (ASF-301) of tHUE-2 cell, human endothelial cell line, and culture medium of these cells (conditioned medium : CM) which affect embryonic development of in vivo fertilized 1-cell embryos of mouse were examined. Two-cell stage block of mouse embryos was overicomed in ASF-301 and CM without EDTA, which usually added in basic medium (modified Whitten Medium: MWM, control) to overcome the 2-cell stage block. The developmental rates of embryos to the blastocyst stage were significantly increased in MWM containing 12.5% of growth factors added to ASF-301 (10mg/ $\ell$ transferrin, 1mg/$\ell$ insulin, 0.01mg/$\ell$ EGF) than those of 100% addition and control, 78.0% vs 20.8 and 52.3%, respectively (P<0.05), but the growth factors was not affected the hatching rate of blastocyst. Using ASF-301 or CM which was not treated, embryonic development into the blastocyst and hatched blastocyst stages were not affected. However, proportions of embryonic development into the blastocyst and hatched blastocyst stages were significantly higher in dilution (ASF-301 1:10; CM 1:3~1:6) than those in control (P,0.05). In ASF-301 dialyzed M.W.<10000 dialysis membrane, the developmental rate upto the hatched blastocyst stage was significantly increased, compared to ASF-301 which was not dialyzed (P<0.05), and hatching rate of blastocyst of these group was singnificantly increased than those in MWM (P<0.05). Compared to CM which was not dialyzed, however, in dialyzed CM was significantly decreased, compared to untreated CM (P<0.05), especially any hatched blastocyst was not appeared. As a result of these experiments indicated that a kind or porper treatment such as a dilution of complex synthetic cell culture medium and conditioned medium, and that a optimal concentration of growth factors are usuful for embryo cultrue in vitro.

• 한국임상수의학회지
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• 제19권1호
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• pp.73-79
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• 2002

To investigate the usability of frozen canine embryos for embryo transfer in the dog, 19 donors, 3 recipients, and 6 male dogs were used for the experiment. Natural mating or artificial insemination was performed for breeding the bitches in natural estrus. Vaginal smear test along with progesterone titre test were performed to detect the appropriate mating time and the bitches were bred twice during 3-6days following LH surge. Embryo collection was done on 8, 9-11, 12-13 days after the second mating to collect morula and blastocyst. Embryos were frozen using a programmable freezer and preseued in LNE tank. Embryos were thawed in 37$^{\circ}C$ water for 15 seconds and transferred into each uterine horn within 30 minutes. Embryos were collected from 13 bitches of 19 donors(68.4%) and the collected embryos were from between 9 and 13 days after 2nd mating. Embryos were produced both by natural mating(60.0%, 9115) and AI with frozen semen(100.0%, 4/4). Embryos were collected from the donors weighed between 2.5 and 30 kg and their age was from 1.5 to 3 years. 52 embryos were collected from 13 donors and the mean number of embryos was four. The stage of embryos was from 2-cell to gastrula and morulae were colledted mostly from 10 to 11 days after 2nd mating. Embryos were collected evenly from each uterine horn and the rate of embryo collection for the number of corpus luteum was 83.9%. Embryos were transferred to 3 recipients(morula 8, blastocyst 1, gastrula 8), however, no offspring was produced.

• 한국가축번식학회지
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• 제26권4호
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• pp.385-394
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• 2002

This study was constructed the correlations of the embryonic developmental rates and the frequency of chromosome aberration using ear-skin-fibroblast cell in nuclear transfer (NT) derived embryos. Karyoplast-oocyte complexes were fused and activated simultaneously, then cultured for seven days to assess development. The developmental rates of NT and in vitro fertilization (IVF) embryos were 55.4% vs 63.5%, 31.7% vs 33% and 13.4% vs 16.8% in 2 cell, 8 cell and blastocyst, respectively. Firstly, the frequency of chromosome aberrations were evaluated using fluorescent in situ hybridization (FISH) technique with porcine chromosome 1 submetacentric specific probe. Chromosome aberration was detected at day 3 on the embryo culture, the percentages of chromosomal aneuploidy in NT and IVF embryos at 4-cell stage were 40%, 31.3%, respectively. Secondly, embryonic fragmentation was evaluated at 4-cell stage embryo. Frequency of embryonic fragmentations was in 51.3% of NT, 61.3% of IVF, 28.9% of parthenogenetic activation at 4-cell stage. The proportion of fragmentation in NT embryos was higher than activation embryos. This result indicates that chromosomal abnormalities and embryonic fragments are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT related with lower implantation rate, increased abortion rate and production of abnormal fetuses.

• 한국수정란이식학회지
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• 제23권1호
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• pp.31-36
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• 2008

We attempted to control the maturation promoting factors (MPF) activity and nuclear remodeling of somatic cell nuclear transfer (NT) bovine embryos. Bovine ear skin fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h and activated. The nuclear remodeling type of the reconstituted embryos was evaluated 1.5 h after activation. MPF activity was assessed in enucleated and chemical treated oocytes before the injection of a donor cell. Effect of chemicals on the embryonic development was evaluated with parthenogenetic embryos. MPF activity increased significantly by caffeine treatment, but decreased by vanadate treatment (p<0.05). Caffeine or vanadate had no deleterious effect on the parthenogenetic embryo development. In caffeine treated group, premature chromosome condensation (PCC) was occurred in 72.2% of NT embryos (p<0.05). In contrast, vanadate induced the formation of a pronucleus-like structure (PN) in a high frequency (68.9%, p<0.05) without PCC (NPCC). Blastocyst development of NT embryos increased by treating with caffeine (30.3%), whereas decreased by treating with vanadate (11.4%) compared to control (22.1%, p<0.05). The results indicate that caffeine or vanadate can control of MPF activity and remodeling type of NT embryos, resulting in the increased or decreased in vitro development.