In this study, the production of transgenic embryo was attempted by microinjection or round spermatid cultured with foreign DNA. At first, the expression of haploid spermatids specific gene, mTP1 in mouse and hPrm2 in hamster spermatids were investigated by RT-PCR method in testes of young mice and hamster testis. The specific gene expression first appeared at 18 days post partum (dpp) in mice spermatid and 20 dpp in hamster spermatid. Therefore, the round spermatids isolated from 17 dpp mice and 19 dpp hamster were used for the introduction of foreign EGFP gene into haploid round spermatids. For the introduction of EGFP gene haploid round spermatids suspended in medium including EGFP gene were treated with a different electric field strength at 0.11, 0.18 and 0.44 ㎸/cm. After electrical stimulation, viability of testicular sperm cells and 67.6%, 66.4% and 49.9%, in mice and 62.6%, 57.9% and 27% in hamster, respectively. These values were significantly lower than those of non-treated control groups 80.5% in mouse and 69.1% in hamster After 72 hrs culture, the highest expression rate of EGFP gene, 28.5% in mice and 32.1% in hamster were obtained from tile spermatogenic cells electroporated by the field strength or 0.18 ㎸/cm. Then, the ability of fertilization and embryonic development of haploid spermatids transfected with foreign EGFP gene were estimated by the microinjection of spermatids into hamster oocytes. The Irate pronuclear formation rate (77.5%) was lower than non-treated control (80%), and the cleavage rate of the treated group (58.8%) was lower than control (65%). To prove the foreign EGFP integration in hamster embryos, 2-cell stage hamster embryos were subjected to the observation under the fluorescence microscope, and the PCR analysis. As a result, about 44% of 2-cell embryos were showed the integration of EFGP gene into their genome. Therefore, These results suggest the possibility to produce transgenic hamsters by microinjection of haploid spermatid transfected with foreign DNA.
Purpose of the present study was to find the optimal ovulation induction medicine for the maturation and development of immature oocytes and culture media for 2-cell embryos in the mouse model. ICR female mouse aged 6 to 8 weeks, were stimulated with 5 IU PMSG injection. At 47 to 50 hour post-PMSG injection, ovaries were dissected out and oocytes-cumulus complexes were punctured. The oocyte-cumulus complexes were cultured in media containing various ovulation induction medicine, CC, HMG and Metrodin for 18 hours. Female ICR mice were stimulated with 5 IU PMSG and 48 hours later were injected 5 IU of hCG, then female and male mice were mated. At 48 hour post-hCG injection, oviducts were dissected out and 2-cell embryos were flushed. The 2-cell embryos were cultured in various media, Ham's F-10 media of milli-Q water $(3^{\circ})$, Ham's F-10 media of HPLC (high performance liquid chromatography, Baxter) water, Medicult media, HTF (human tubal fluid) media for 96 hours. The results were as follows. 1. When the oocytes-cumulus complexes were cultured in $10^{-9}{\mu}g/ml{\sim}10^{-8}{\mu}g/ml$ of CC, those were suppressed in meiotic maturation $(28.2{\sim}33.7%)$. Whereas the oocytes-cumulus complexes were cultured in $10^{-7}{\mu}g/ml{\sim}10^{-4}{\mu}g/ml$, these were not effected in meiotic maturation $(54.5{\sim}72.7%)$. 2. When the oocytes-cumulus complexes were cultured in $10^{-4}{\mu}g/ml{\sim}10^{-1}{\mu}g/ml$ of Metrodin, those were suppressed in meiotic maturation $(35.7{\sim}41.5%)$. Meanwhile the oocytes-cumulus complexes were cultured in $10^{-7}{\mu}g/ml{\sim}10^{-5}{\mu}g/ml$, those were not effected in meiotic maturation $(54.2{\sim}70.3%)$. 3. When the oocytes-cumulus complexes were cultured in $10^{-5}{\mu}g/ml{\sim}10^{-4}{\mu}g/ml$ of HMG, those were suppressed in meiotic maturation $(48.2{\sim}50.4%)$. As being cultured in $10^{-7}{\mu}g/ml{\sim}10^{-6}{\mu}g/ml$, increased in meiotic maturation $(75.8{\sim}80.7%)$. 4. When the 2-cell embryos were cultured in Ham's F-10 media of milli-Q water $(3^{\circ})$, Ham's F-10 media of HPLC (high performance liquid chromatograpy, Baxter) water, Medicult media, HTF (human tubal fluid) media, developmental rates to blastocyst and hatching for 96 hour were 50.0%, 45.2%, 71.5% and 95.6%, respectively.
This study was conducted to investigate the effect of embryo stage on bisection rates of embryos, on development of separated demi-embryos and on subsequent development to full term following transfer of demi-embryos to recipients. The results obtained in this study are summarized as follows. The 2-, 4-, 8-cell and morula embryos were mostly obtained at 36-42, 48-54, 62-66 and 72-78 hours after injection of hCG, respectively, and the average number of embryos recovered per head was 17.0. The bisection rate of 8-cell embryos was 83.6%, which was significantly higher than that of morula embryos, 65.5%. But the development of morula demi-embryos to blastocyst after in-vitro culture was better than that of 8-cell demi-embryos and the rate was 76.5% and 60.9%, respectively. When the 2, 10 and 15 cultured demi-embryos or intact embryos were transferred to each recipient mouse, respectively. the highest pregnancy rate was obtained when 15 embryos were transferred. And the overall offspring production rate of intact embryos was higher than that of demi-embryos.
Journal of the Korean Society of Clothing and Textiles
/
v.24
no.8
/
pp.1146-1154
/
2000
The purpose of this study was to investigate the antimicrobial activity, antimutagenic and anticancer effects and dyeing properties of the fermented indigo extract. The methanol extract of fermented indigo showed a strong inhibition effect on Trich. mentagrophytes and antimutagenic activities against aflatoxin B$_1$(AFB$_1$) in the Ames test using Salmonella typhimurium TA 100. We also found in vitro anticancer effects of the methanol extract of fermented indigo and it was evaluated by using Clone M-3 mouse melanoma cells and A431 human epidermoid carcinoma cells and exerted little cytotoxity against 3T3-L1 embryo fibroblast cell. In the relationship between the K/S values of cotton and silk fabrics dyed with fermented indigo and dyeing repeating times, the K/S values became higher as the repeating times were increased. The K/S values were high when the fabrics were dyed at low temperture. The K/S values of cotton fabrics were higher than those of silk fabrics. Changes of surface color of silk fabrics were higher than those of cotton fabrics after water fastness test, laundering, irradiation and treatment of acidic and alkaline perspiration.
Kang M. Y.;Han M. S.;Lee S. C.;Kim J. H.;Sohn S. H.
Reproductive and Developmental Biology
/
v.29
no.1
/
pp.1-7
/
2005
Telomeres consisting of (TTAGGG)n tandem repeat DNA sequences and associated proteins are essential for chromosome stability and related with cell senescence, apoptosis and cancer. The telomerase is a ribonucleoprotein which act as a template for the synthesis of telomeric DNA. This study was carried out to identify the distribution of telomeres on mouse chromosomes and also to analyze the amount of telomeres and telomerase activity of mouse embryos at early embryonic stages. Germ cells and early embryos from 1 cell to blastocyst stage were analyzed. The amount of telomeres was analyzed by quantitative fluorescence in situ hybridization technique(Q-FISH) using a human telomeric DNA probe, and telomerase activity was measured by telomeric repeat amplification protocol assay(TRAP). In results, the telomeres on mouse chromosomes were distributed at the ends of all autosomes and sex chromosomes. Although the quantity of telomeres varied among chromosomes, most of chromosomes had higher amount in q-arm telomeres than in p-arm telomeres. The results of Q-FISH indicated that the relative amount of telomeres of mouse embryos in each embryonic stage was approximately the same except the higher amount in blastocysts. Using TRAP assay on mouse embryos, telomerase activity was detected in all preimplantation stages from mature oocytes to blastocysts. Especially the telomerase activity was significantly increased at the morula and blastocyst stage. In conclusion, there may be a close association between the amount of telomeres and telomerase activity in early embryonic stages, and analysis of telomere quantity and telomerase activity on early development will be helpful for the investigation of embryogenesis and embryonic cell differentiation in mice.
Kang D.W.;Hur C.G.;Choi C.R.;Park J.Y.;Hong S.G.;Han J.H.
Journal of Embryo Transfer
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v.21
no.1
/
pp.35-43
/
2006
Ions play important roles in various cellular processes including fertilization and differentiation. However, it is little known whether how ions are regulated during early embryonic development in mammalian animals. In this study, we examined changes in $Ca^{2+}\;and\;K^+$ concentrations in embryos and oviduct during mouse early embryonic development using patch clamp technique and confocal laser scanning microscopy. The intracellular calcium concentration in each stage embryos did not markedly change. At 56h afier hCG injection when 8-cell embryos could be Isolated from oviduct, $K^+$ concentration in oviduct increased by 26% compared with that at 14h after injection of hCG During early embryonic development, membrane potential was depolarized (from -38 mV to -16 mV), and $Ca^{2+}$ currents decreased, indicating that some $K^+$ channel might control membrane potential in oocytes. To record the changes in membrane potential induced by influx of $Ca^{2+}$ in mouse oocytes, we applied 5 mM $Ca^{2+}$ to the bath solution. The membrane potential transiently hyperpolarized and then recovered. In order to classify $K^+$ channels that cause hyperpolarization, we first applied TEA and apamin, general $K^+$ channel blockers, to the bath solution. Interestingly, the hyperpolarization of membrane potential still appeared in oocytes pretreated with TEA and apamin. This result suggest that the $K^+$ channel that induces hyperpolarization could belong to another $K^+$ channel such as two-pore domain $K^+(K_{2P})$channel that a.e insensitive to TEA and apamin. From these results, we suggest that the changes in $Ca^{2+}\;and\;K^+$ concentrations play a critical role in cell proliferation, differentiation and reproduction as well as early embryonic development, and $K_{2P}$ channels could be involved in regulation of membrane potential in ovulated oocytes.
Ji, Min-Young;Lee, Young-Choon;Kim, Kyoung-Sook;Cho, Jin-Won;Jung, Kyu-Yong;Kim, Cheorl-Ho;Choo, Young-Kug
Archives of Pharmacal Research
/
v.22
no.3
/
pp.243-248
/
1999
Sialic acids are key determinants for biological processes, such as cell-cell interaction and differentiation. Sialyltransferases contribute to the diversity in carbohydrate structure through their attachment of sialic acid in various terminal positions on glycolipid and glycoprotein (N-linked and O-linked) carbohydrate groups. Gal$\beta$ 1,3(4)GlcNAc $\alpha$2,3-sialyltransferase (ST3Gal III) is involved in the biosynthesis of $sLe^{X}$ and sLe^{a}$ known as selection ligands and tumor-associated carbohydrate structures. The appearance and differential distribution of ST3Gal III mRNA during mice embryogenesis [embryonic (E) days; E9, E11, E13, E15] were investigated by in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. On E9, all tissues were positive for ST3Gal III mRNA expression whereas ST3Gal III mRNA on E11 was not detected throughout all tissues. On E13, ST3GAl III mRNA was expressed in different manner in various tissues. In this stage, ST3Gal III mRNA was positive only in the liver, pancreas and bladder. On E15, specific signal for ST3GAl III was detected in the liver, lung and forebrain. These results indicate that ST3Gal III is differently expressed at developmental stages of mice embryo, and this may be importantly related with regulation of organogenesis in mice.
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.
Background: Ginsenoside Rh2 is well known for many pharmacological activities, such as anticancer, antidiabetes, antiinflammatory, and antiobesity properties. Glycosyltransferases (GTs) are ubiquitous enzymes present in nature and are widely used for the synthesis of oligosaccharides, polysaccharides, glycoconjugates, and novel derivatives. We aimed to synthesize new ginsenosides from Rh2 using the recombinant GT enzyme and investigate its cytotoxicity with diverse cell lines. Methods: We have used a GT gene with 1,224-bp gene sequence cloned from Lactobacillus rhamnosus (LRGT) and then expressed in Escherichia coli BL21 (DE3). The recombinant GT protein was purified and demonstrated to transform Rh2 into two novel ginsenosides, and they were characterized by nuclear magnetic resonance (NMR) techniques and evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay. Results: Two novel ginsenosides with an additional glucopyranosyl (6→1) and two additional glucopyranosyl (6→1) linked with the C-3 position of the substrate Rh2 were synthesized, respectively. Cell viability assay in the lung cancer (A549) cell line showed that glucosyl ginsenoside Rh2 inhibited cell viability more potently than ginsenoside Rg3 and Rh2 at a concentration of 10 μM. Furthermore, glucosyl ginsenoside Rh2 did not exhibit any cytotoxic effect in murine macrophage cells (RAW264.7), mouse embryo fibroblasts cells (3T3-L1), and skin cells (B16BL6) at a concentration of 10 μM compared with ginsenoside Rh2 and Rg3. Conclusion: This is the first report on the synthesis of two novel ginsenosides, namely, glucosyl ginsenoside Rh2 and diglucosyl ginsenoside Rh2 from Rh2 by using recombinant GT isolated from L. rhamnosus. Moreover, diglucosyl ginsenoside Rh2 might be a new candidate for treatment of inflammation, obesity, and skin whiting, and especially for anticancer.
Park H.;Kim J. Y.;Kim J. Y.;Lee J. H.;Park H. D.;Kim J. M.
Journal of Embryo Transfer
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v.19
no.3
/
pp.245-255
/
2004
In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2% versus 15.4%, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0% versus 53.9%, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.
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