• Title/Summary/Keyword: 2-cell embryo

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Cloning and Characterization of Homeodomain-Zip Gene, Phc5 in Embryogenic Callus derived from Pimpinella brachycarpa Suspension Cultured Cells (참나물 현탁배양세포 유래 배발생캘러스에서 HD-Zip 유전자, Phc5의 클로닝과 특성)

  • 손수인;김준철
    • Korean Journal of Plant Tissue Culture
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    • v.26 no.2
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    • pp.121-126
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    • 1999
  • Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.

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Ginseng Transformation of Betaine Aldehyde Dehydrogenase Gene Relative Salt Resistant through Somatic Embryogenesis (염류내성관련 유전자 Betaine Aldehyde Dehydrogenase Gene의 인삼 체세포 배발생을 통한 형질전환)

  • Yoon Young-Sang;Bae Chang-Hyu;Song Won-Seob;Yoon Jae-Ho;Yang Deok-Chun
    • Korean Journal of Plant Resources
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    • v.18 no.1
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    • pp.15-21
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    • 2005
  • Korean ginseng(Panax ginseng C.A. Meyer) is very difficult to obtain stable production of qualified ginseng roots because of variable stresses in soil environments. In transformation of ginseng with betain aldehyde dehydrogenase gene, compounds synthesized for controlling osmotic pressure such as proline, glycine, betaine, polyols and sugar were accumulated in cell for salt resistance in transgenic plants. 2 Agrobactgerium conjugants were acquired with bet A and bet B genes for solt resistant plants. A. tumefaciens MP90/pBetA and A. tumefaciens MP90/pBetB were recombined for increasing the tolerance to salt stress. To confirm the transformation of the binary vector, tobacco plant was transformed, and the transformant can grow on media containing high concentration of kanamycin. To identify NPT 11, BetA and BetB genes of the transformants, the band on the agarose was confirmed by PCR and RT-PCR techniques. The transformants of ginseng with bet A and bet B genes were acquired on the phytohormone free basic MS media containing only antibiotics and 1M mannitol used for selection of transgenic plant, but the transfomation efficiency for BetA and BetB was very low.

Sex Determination in Somatic and Embryonic Cells of the Pig by FISH and PCR (FISH와 PCR에 의한 돼지 체세포 및 배아세포의 성 판정)

  • Chung, Y.;Jeon, J.T.;Kim, K.D.;Lee, S.H.;Hong, K.C.
    • Korean Journal of Animal Reproduction
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    • v.20 no.3
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    • pp.323-331
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    • 1996
  • Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

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STUDIES ON THE DIMORPHISM AND TRANSITION OF BISEXUALITY OF HETEROSTYLOUS POLYGONACEAE (여뀌과 이형경식물의 Dinorphism과 Bisexuality의 변화)

  • Harn, Chang-Yawl
    • Journal of Plant Biology
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    • v.3 no.2
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    • pp.6-18
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    • 1960
  • The present experiments were designed in order to clarify the differences between the long and short styled plants and the transgressive gradition in the degree of dimorphism among the three heterostylous species of the Polygonus, P. japonica, F. esculentum, and P. senticosa, based on investigations regarding the floral structure, ecological and physiological traits, the results of which are summarized as follows: (1) P. japonica, although it exhibits typical dimorphism, has undergone so high a differentiation between long and short styled that its long styled individuals behave as if they were female; and short styled individuals as if male. In long-styled individuals, filament, anther, and pollen grains show signs of degeneration, most of the pollen being abortive. On the other hand, in short styled individuals, the filament, anther, and pollen grains have attained remarkable development; the pollen grians are large and fertile. In short-plant the fertilized flowers readily drop off in every stage of their embryo development. This species has completely lost the self-fertile property, which is characteristic of the non-dimorphic Polygonum genus. Although this specsei typically exhibits the physiological characteristics of the non-dimorphic Polygonum genus. Although this specisei typically exhibits the physiological characteristics of dimorphism in controlled pollination, the short-styled individuals bear no seed in nature, thus misleading taxonomists to idenfity the short-styled plant as male. 2) The morphological feature of the flower organ of P. senticosa obviously indicates definite dimorphism. Physiologically, however, no differentiation towards dimorphism was observed, the species still retaining, both in long and short-individuals, the self-fertile property common to the Polygonum genus. Elaborate examinations revealed that regardless of the modes of pollination, both fertiization and seed setting flourish, no differentiation betwen legitimate and illegitimate unions being recognizable. This sort of physiological property has not been observed in the investigations of other heterostylous plants. It is assumed that this species is differentiated structurally into dimorphism, but not yet physiologically. In nature, however, this plant would have more opportunities to be cross-pollinated, i.e., legitimately combined, than self-pollinated because of the development of two forms of flowers. 3) In terms of heterostylism, the F. esculentum just occupies the intermediate position between P. japonica and P. senticosa structurally, ecologically, and physiologically. Doescription of some of the physiological behavior of the plant will suffice to demonstrate the above facts. While P. japonica has completely lost its self-fertile property, P. senticosa still retains it wolly. In F. esculentum 2-6% of self-fertility is the result in illegitimate combination. There occur occasionally hereditary self fertile individuals among some of the F. or 20 min. irradiation plot, when they reach any stage of the same bacterial population. In addition to this increase of total population in the plots with the more dose of UV light irradiation, it seems that the more dose of UV light irradiation is the more shortened the generation time of Azotobacter. Therefore, it is clear that variation of reproductive rate must be, mere or less, due to the genetic effects induced by UV light irradiation. On the other hand, the lag phase or logarithmic growth phase in nonirradiated culture is shortened prominently, and this must be due to the difference in bacterial number of the original inoculm. The generation time of Azotobacter is shortened by exogeneous treatment of nuclei acid derivatives, and the degree is greater in case of DNA derivatives than RNA dervatives. W.H. Price reported that the rate of ribose nucleic acid to protein in Staphylococcus muscae is proportional to the generation time: that is the faster the cell can form ribose nucleic acid, the more rapid its growth. This explains the shortening of generation time by exogeneous RNA derivatives in this work reasonably. On the other hand, it is well known that the desoxyribose nuclic acid content per cell is constant and independent of the generation time. A.D. Laren and W.N. Takahashi reported that the infectious RNA from TMV is 6 times as sensitive to inactivation by UV as it is in the form of intact virus, and that inactivation of infectious TMV involves onlu a local change on RNA chain. But, the effect of exogeneous DNA in this work suggests that irradiated living cell which cotain DNA bring about some change on DNA moleculs as well as RNA molecules. And if the mutagenic effects of UV take into consideration, it is very reasonable. Therefore, it is clear that the variation of the generation time by UV irradiation is, more or less, due to the genetic effects. Therefore, it seems that the shortness of the average lifewpan of Azotobacter by UV irradiation is resulted not only from the influence of the environmental conditions, but also from the variation of genetic factor of the individual.

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Effect of Fragment Removal on Development of Human Fragmented Embryos in IVF-ET Program (IVF-ET 시술 시 파편제거가 파편화된 인간배아의 발달에 미치는 효과)

  • Chi, Hee-Jun;Koo, Jung-Jin;Lee, Ju-Ok;Ryu, Hyoung-Eun;Kim, Kwang-Rae;Park, Chan;Roh, Sung-Il
    • Clinical and Experimental Reproductive Medicine
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    • v.37 no.4
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    • pp.339-348
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    • 2010
  • Objective: To investigate the beneficial effect of fragment removal on the subsequent cell division and clinical outcome of the fragmented human embryos. Methods: A prospective study was performed in Hanna Women's Clinic and Mizmedi Hospital. Sixty couples undergoing In vitro fertilization-embryo transfer (IVF-ET) program were participated in the present study. The microsurgical fragment removal was performed in 106 fragmented embryos of 29 patients before the transfer. As a control group, 122 fragmented embryos of 31 patients were transferred without the fragment removal. Effects of fragment removal on morphological changes and clinical outcomes of fragmented embryos were investigated. Results: Mean morphological grade (G2.79) of fragmented embryos was significantly improved after the fragment removal(G1.63, p<0.001). Most of the fragmented embryos did not show a regeneration of fragments after the fragment removal during the subsequent development, and a beneficial effect of fragment removal on the development of the fragment removed embryos was observed. Implantation and pregnancy rates of fragment removed embryos were 12.3% and 31.3%, whereas the rates of control group embryos were 6.6% and 22.5%, respectively. There was no statistical significance in the rates between the two groups because of the low number of trials. Conclusion: Microsurgical fragment removal improved the subsequent development as well as the morphological grade of fragmented embryos. The fragment removal may be beneficial for neighboring blastomeres by repairing the intercellular communication and removing the secretion of the potential toxic materials by fragments.

Superovulation-Oocyte and Uterine Function (과배란-난자 및 자궁기능)

  • 문영석
    • Korean Journal of Animal Reproduction
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    • v.20 no.4
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    • pp.379-384
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    • 1997
  • Superovulation with exogenous gonadotropins creates a spectrum of pre or periovulatory hormonal changes with subsequent detrimental effects on oocyte quality, fertilization, embryo development, implantation and maintenance of pregnancy. Our recent study determined potential roles for insulin-like growth factor-1 (IGF-1) in uterine environment regulation and preimplant tation in the rat. The evidence indicates that IGF-l may play an important role in the main tenance of a receptive uterine environment for embryonic development and the regulation of decidualization. Embryonic loss and failure of implantations following superovulation may be partially attributed to disturbances in uterine IGF-l action as observed in this study. We investigated the effects of superovulatory doses of gonadotropins on frequency of chromosomal a abnormalities of mouse embryos. Chromosome a analysis of mouse zygotes and 8- to 16-cell stage embryos from spontaneously ovulated, 5, 10, and l 15 lU pregnant mare serum gonadotropin (PMSG) superovulated mice was carried out. Aneuploidy, polyploidy and structural chrom- osomal abnormalities were detected among the four groups. However, only polyploidy was correlated with superovulation. In 10 and 15 IV PMSG treated groups, the rate of polypoidy was 2.9% and 10.5%, respectively. Furthermore, there was a dose reponse relationship between the PMSG dose and the incidence of embryonic p polyploidy (P

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Toxic Effects of Arsenic and Chromium on the Fertilization and Embryo Development Rates in the Sea Urchin (Hemicentrotus pulcherrimus) (말똥성게(Hemicentrotus pulcherrimus)의 수정 및 배아 발생률에 대한 비소와 크롬의 독성영향)

  • Hwang, Un-Ki;Ryu, Hyang-Mi;Yu, Jun;Kang, Han-Seung
    • Korean Journal of Environmental Biology
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    • v.31 no.2
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    • pp.69-77
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    • 2013
  • Toxic effects of arsenic (As) and chromium (Cr) has been investigated using the sea urchin (Hemicentrotus pulcherrimus) germ cell and pluteus-larvae. The gametotoxic and embryotoxic effects of As and Cr on H. plucherrimus were each investigated at 6.25, 12.5, 25, 50, 100. Spawning was induced by 0.5 M KCl solution and the normal fertilization and embryogenesis rates were performed for 10 min and 64 hrs after fertilization, respectively. The normal fertilization and embryogenesis rates in the control condition (not including As and Cr) were greater than 94% and 93%, respectively. The fertilization rate was not significantly changed compared with control but embryogenesis rate was significantly decreased with concentration-dependent manner. As and Cr reduced normal embryogenesis rates and a significant reduction occurred at concentration greater than 6.25 ppb (P<0.01) and 25 ppb (P<0.05), respectively. The lowest-observedeffect- concentration (LOEC) of normal embryogenesis rate in As and Cr were each 6.25 and 25 ppb, respectively. From these results, normal embryogenesis rate of H. pulcherrimus have toxic effect at greater than the 6.25 ppb concentration of As and 25 ppb concentration of Cr in marine ecosystems. These results suggest that the normal embryogenesis rates of H. pulcherrimus are very useful test method for the toxicity assessment of heavy metal as As and Cr in marine ecosystems.

Gene Expression Profiling of Oilseed Rape Embryos Using Microarray Analysis (Microarray 분석을 이용한 유채 종자성숙단계별 유전자 발현 양상)

  • Roh, Kyung Hee;Park, Jong-Sug;Kim, Jong-Bum;Kim, Hyun Uk;Lee, Kyeong-Ryeol;Kim, Sun Hee
    • Journal of Applied Biological Chemistry
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    • v.55 no.4
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    • pp.227-234
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    • 2012
  • We observed that oil began to accumulate at 25 seed days after flowering (DAF) and reached the maximum potential at 35 seed DAF of oilseed rape, and the greatest weight of 100 seeds was obtained at 35 seed DAF. To survey a broad analysis of gene expression in developing embryos of Brassica napus, the Bn 300k microarray have been constructed. The Bn 300k Microarrary was designed from 80,696 unigenes clustered from 543,448 ESTs and 780 cDNA at NCBI. These arrays have been hybridized in a series of experiments with probes derived from seeds and leaf of B. napus. Approximately 8.5% of the 7,000 genes were expressed as ratios 2-fold higher in seed (25 DAF) than leaves and 0.4% at ratios 10. Also we observed that storage and cell differentiation-related genes were highly expressed at 10 DAF, whereas energy-related genes including fatty acid metabolism were increased up depending on seed maturation using Microarray, which was confirmed by reverse transcriptase polymerase chain reaction. These results suggest that B. napus arrays provide a very useful data set of seed-specific expression that can be further analyzed by examination of the promoter regions of these genes and help our understanding of the complex regulatory network in developing seeds.

Effects of Heparin-Binding Epidermal Growth Factor on the Expression of MMP-9 and ATPase ${\gamma}$-subunit mRNA in the Mouse Embryo (생쥐 배아에서 Heparin-Binding Epidermal Growth Factor가 MMP-9과 ATPase ${\gamma}$-subunit 유전자의 발현에 미치는 영향)

  • Hong, Seok-Ho;Nah, Hee-Young;Lee, Ji-Yun;Kim, Jung-Hee;Kim, Chung-Hoon;Chae, Hee-Dong;Kang, Byung-Moon;Kim, Moon-Kyoo
    • Clinical and Experimental Reproductive Medicine
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    • v.28 no.2
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    • pp.87-93
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    • 2001
  • Objectives: The purpose of this study was to evaluate effects of heparin-binding epidermal growth factor (HB-EGF) on the rate of blastocyst formation and hatching in the mouse embryos and the expression of matrix metalloproteinase-9 (MMP-9) and ATPase ${\gamma}$-subunit mRNA. Methods: Late 2-cell mouse embryos was cultured for 72 hours in RTF medium containing with 1, 10, and 100 ng/ml HB-EGF. The mRNA expression level of MMP-9 and ATPase ${\gamma}$-subunit was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results: The rate of hatching was significantly higher (p<0.05) in group containing with 1 ng/ml HB-EGF than other groups. Also, the rate of hatched blastocyst was significantly higher (p<0.05) in 10 ng/ml. The mRNA expression level of MMP-9 mRNA was not shown any difference among groups, but ATPase ${\gamma}$-subunit was higher than other groups. Conclusions: Taken together these results suggest that HB-EGF has the positive effect to promote the blastocyst formation and hatching process and influences the blastocoel expansion by promoting the ATPase mRNA expression in the mouse embryos.

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The Efficacy of Recombinant Human Follicle Stimulating Hormone (rhFSH) in Human IVF-ET Program (체외수정시술시 유전자 재조합 난포자극호르몬제의 효용성)

  • Han, Kuk-Sun;Lee, Hong-Bok;Song, In-Ok;Park, Yong-Seog;Byun, Hye-Kyung;Jun, Jin-Hyun;Koong, Mi-Kyoung
    • Clinical and Experimental Reproductive Medicine
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    • v.29 no.1
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    • pp.45-56
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    • 2002
  • Objectives: Recently, recombinant FSH (rFSH) has been manufactured using a Chinese hamster ovary cell line transfected with the gene encoding human FSH. Both rFSH and urinary gonadotropin (uFSH) could be used for controlled ovarian hyperstimulation (COH). However, uFSH implies a number of disadvantages, such as batch-to-batch inconsistency, no absolute source control, dependence on large amounts of urine, low specific activity, and low purity. The purpose of this study was to evaluate the efficacy of rFSH in human IVF-ET program. Materials and Methods: A total of 508 infertile women was enrolled in this study. They are classified into rFSH group (n=177) or uFSH group (n=331), and all of them were matched by age and cause of infertility in same period. The $Puregon^{(R)}$ (Organon, Holland) was used as rFSH, and the Metrodin-$HP^{(R)}$ (Serono, Switzeland) and $Humegon^{(R)}$ (Organon, Holland) was used as uFSH. We subdivided the patients into three age groups. The outcomes of IVF-ET program were analyzed using the statistical package for social sciences (SPSS). Results: There was no significant differences in the level of estradiol on hCG injection day, the numbers of retrieved oocytes, matured oocytes, fertilized oocytes, transferred embryos, frozen embryos between the two groups. The total dose (IU) of gonadotropin for COH was significantly lower in the rFSH group compared to uFSH group ($1339{\pm}5491.1$ vs $2527.8{\pm}1075.2$ IU, p<0.001). Clinical pregnancy rate per embryo transfer in the rFSH group showed increasing tendency, compared to the uFSH group, but there was no statistical significance (35.2% vs 29.3%). Our results demonstrated that the relative efficiency of rFSH compared with uFSH is higher in older patients. Conclusions: The ovarian stimulatory effect and clinical outcome of recombinant FSH was similar to that of the urinary gonadotropin. The IVF-ET cycles with significantly lower dose of gonadotropin in rFSH group showed comparable results. Therefore, we suggest that recombinant FSH is more potent and effective than urinary gonadotropin.