Biomedical subjects

So Hyun Lee

Publications and source records attributed to So Hyun Lee.

13 recordsLinked to original sources

PubMed2006

Improved in vitro bovine embryo development and increased efficiency in producing viable calves using defined media.

In this study, we developed a defined culture medium that supported improved in vitro bovine embryo development and calving rate after embryo transfer (ET). In vitro-matured bovine oocytes from abbatoir-derived ovaries from Korean native, HanWoo cattle were fertilized with frozen-thawed spermatozoa and embryos were cultured in two-step culture media. In Experiment 1, embryos were cultured in media supplemented with 8 mg/mL BSA, or 0.1mg/mL PVA and 8 mg/mL BSA+2.77 mM myo-inositol or 0.1mg/mL PVA+2.77 mM myo-inositol. Although defined culture media containing PVA supported lower developmental competence compared to undefined media (containing BSA; 8% versus 34%, respectively), defined culture media containing 2.77 mM myo-inositol increased rates of blastocyst formation up to 28%. In Experiment 2, the effect of energy substrate (1.5mM glucose or 1.2mM phosphate) in PVA-myo-inositol defined culture medium on in vitro embryo development was investigated. Defined culture media containing PVA, myo-inositol and phosphate supported better embryo development to blastocysts compared to medium supplemented with both glucose and phosphate (43% versus 31%). In Experiment 3, the effect of epidermal growth factor (EGF) in PVA+myo-inositol-phosphate two-step culture medium on in vitro embryo development was investigated. Among 0, 1, 10 and 100 ng/mL EGF concentrations, the maximal effect was observed with 10 ng/mL EGF (52% blastocyst formation). In Experiment 4, total cell number and calving rate were compared between defined PVA-myo-inositol-phosphate-EGF medium and undefined medium containing BSA, glucose and phosphate. No differences in total cell number of blastocysts obtained from the two groups were observed, however, the rate of viable offspring production was increased using the defined culture medium, compared to the undefined culture medium. In Experiment 5, the relative abundance of mRNA transcripts [interferon-tau (If-tau), glucose transporter-1 (glut-1) and insulin like growth factor 2 receptor (Igf2r)] were analyzed in blastocysts derived from undefined or defined culture media. Gene expression of If-tau, glut-1 was significantly increased in defined culture medium compared to undefined medium. In conclusion, chemically defined culture media without BSA or FBS improved developmental competence of in vitro cultured bovine embryos and delivery of viable calves after ET.

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PubMed2006

Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes.

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P<0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones.

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PubMed2006

Antiapoptotic and embryotrophic effects of alpha-tocopherol and L-ascorbic acid on porcine embryos derived from in vitro fertilization and somatic cell nuclear transfer.

The objective was to determine optimal concentrations of alpha-tocopherol and l-ascorbic acid for development of porcine embryos derived from in vitro-fertilization (IVF) or somatic cell nuclear transfer (SCNT). The frequency of blastocyst formation in IVF embryos was 17.6, 28.6, 32.4 and 21.4% for control, 50, 100 and 200microM alpha-tocopherol, respectively, whereas in SCNT embryos, the frequency was 12.8, 19.0, 24.8 and 17.7% for corresponding concentrations of alpha-tocopherol. For both IVF and SCNT embryos, there were significantly more cells in blastocysts and the embryos had greater developmental competence when the embryo culture medium was supplemented with 100microM alpha-tocopherol. Although either alpha-tocopherol or l-ascorbic acid reduced the proportion of apoptotic cells in blastocysts, in combination they resulted in rates of apoptosis that were similar to the control group. For IVF embryos, the apoptotic index was 0.09 and 0.11 for alpha-tocopherol or l-ascorbic acid at a concentration of 100microM, respectively. Conversely, when these antioxidants were supplemented together, the apoptotic index increased significantly and was similar to the control group (i.e., 0.17 and 0.21 for combined and control group). For SCNT embryos, the apoptotic index was 0.10 for 100microM for both alpha-tocopherol and l-ascorbic acid, whereas the index was 0.23 and 0.17 for control and combined group. In conclusion, we recommend supplementing porcine embryo culture medium with 100microM alpha-tocopherol or 100microM l-ascorbic (as a second choice).

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PubMed2006

Embryotrophic effects of ethylenediaminetetraacetic acid and hemoglobin on in vitro porcine embryos development.

This study investigated the embryotrophic effects of ethylenediaminetetraacetic acid (EDTA) and hemoglobin (Hb) on porcine preimplantation embryo development. Porcine embryos produced by in vitro maturation/fertilization were cultured for 6 days in modified North Carolina State University-23 medium (mNCSU-23) supplemented with EDTA and/or Hb. In Exp. 1, culturing porcine zygotes with 100 microM EDTA significantly increased cleavage frequencies (85.3%) at 48 h post insemination and the number of inner cell mass (ICM) (9.6+/-5.5) compared to the control (7.0+/-2.8). However, 100 microM EDTA did not improve blastocyst formation compared to 0, 1 or 10 microM EDTA. In Exp. 2, in vitro fertilized oocytes were cultured with 0, 1 or 10 microg/ml Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the cell numbers of blastocysts in 1 microg/ml Hb compared to 0 or 10 microg/ml Hb. In Exp. 3, culturing embryos with 100 microM EDTA+1 microg/ml Hb significantly improved frequencies of cleavage, blastocyst formation, and total cell numbers in blastocysts compared to the control. Moreover, 100 microM EDTA, 1 microg/ml Hb and their combination reduced reactive oxygen species (ROS) accumulation and decreased the incidence of apoptosis. In conclusion, the present study clearly demonstrated that the combining treatment of EDTA and Hb improved IVF porcine embryo development.

Animals↗

PubMed2006

Anti-apoptotic effect of insulin-like growth factor (IGF)-I and its receptor in porcine preimplantation embryos derived from in vitro fertilization and somatic cell nuclear transfer.

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos.

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PubMed2005

Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein.

This study was performed to develop a system for porcine somatic cell nuclear transfer (SCNT) and to produce human erythropoietin (hEPO)-transgenic cloned piglets. Porcine fetal fibroblasts were transfected with an expression plasmid (phEPO-GFP). In Experiment 1, the effect of transfection of phEPO-GFP transgene on development of porcine SCNT embryos was investigated. Three fetal fibroblast cell lines (two male and one female) with or without transfected with phEPO-GFP trasngene were used as donor cells for SCNT. Lower fusion rates were observed in two lines of transfected cells as compared to those of the control cells. In Experiment 2, the effect was examined of elevated Ca2+ concentration in the fusion/activation medium on development of transfected SCNT embryos. The rates of fusion and blastocyst formation were significantly increased by supplementing 1.0 mM of CaCl2 (versus 0.1 mM) into the fusion/activation medium. In Experiment 3, the effect was studied of a chemical treatment (cytochalasin B) after electric fusion/activation (F/A) on porcine transgenic SCNT embryo development. The electric F/A + cytochalasin B treatment increased total cell number in blastocysts as compared to that of electric F/A treatment alone. In Experiment 4, transgenic cloned embryos were transferred to surrogate mothers and a total of six cloned piglets were born. Transgenic cloned piglets were confirmed by polymerase chain reaction and Southern blot analysis. From a single surrogate mother, female and male transgenic cloned piglets were produced by transferring pooled SCNT embryos derived from female and male transfected donor cells. In conclusion, a system for porcine SCNT was developed and led to the successful production of hEPO transgenic cloned piglets.

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PubMed2005

The role of gonadotropin-releasing hormone (GnRH) and its receptor in development of porcine preimplantation embryos derived from in vitro fertilization.

This study was performed to investigate the expression of embryo-derived gonadotropin-releasing hormone (GnRH) and its receptor, and to determine the role of GnRH in porcine preimplantation embryos. In Experiment 1, porcine blastocysts derived from in vitro fertilization (IVF) and cultured in North Carolina State University (NCSU)-23 medium were subjected to reverse transcription polymerase chain reaction (RT-PCR) amplification with specific primers for GnRH and its receptor. The results showed that GnRH and its receptor were expressed in porcine IVF blastocysts. In order to investigate the role of GnRH in embryo development, porcine IVF embryos were cultured in NCSU-23 supplemented with different concentrations (0, 0.1, 1, or 10 microM) of a GnRH agonist (leuprolide, Experiment 2) or GnRH antagonist (antide, Experiment 3). Supplementing the culture medium with 0.1 or 1 microM leuprolide increased the rate of blastocyst formation (28.5 or 27.6% versus 20.2%) and mean total cell number (129 versus 104) compared to the control group. In contrast, antide significantly decreased the rate of blastocyst formation [12.6% (0.1 microM), 10.2% (1.0 microM), or 8.9% (10.0 microM) versus 22.8% (control)] and total cell number [69 (1 microM) or 68 (10 microM) versus 104 (control)]. In Experiment 4, porcine IVF embryos were cultured in NCSU-23 medium containing 1 microM antide plus 1 microM leuprolide. The embryotrophic effect of GnRH agonist was reversed by co-supplementing with GnRH antagonist. In conclusion, the present study demonstrated that supplementing a culture medium with GnRH agonist can improve blastocyst formation and the quality of porcine IVF embryos, and that this action was mediated through GnRH receptors.

Animals↗

PubMed2005

Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation.

A novel porcine cDNA array, containing 1,015 PCR products selected for embryonic expression, was used for transcriptional profiling of conceptuses at four stages of peri-implantation development. Total conceptus RNA from small spherical, large spherical, tubular, and filamentous stages was amplified, converted to cDNA, and hybridized to membranes. Initially, normalized signal intensities obtained using cDNA from total RNA or from amplified RNA were compared. Uniform distribution of P-values associated with t-tests conducted for each gene indicated no evidence that amplification introduced bias. Analysis of data obtained by using amplified targets and the novel array identified genes differentially expressed across stages. Such genes were identified by testing for significant stage effects in gene-specific mixed models. A total of nine genes were declared differentially expressed. Six of the nine genes had P-values less than 0.001, and a false discovery rate of approximately 17% was associated with this significance threshold. Two out of six genes were significant when using the Bonferroni method to control the probability of one or more false positives. The other three genes had P-values between 0.001 and 0.01 and exhibited differences greater than twofold between stages. All four genes selected for confirmation (steroidogenic acute regulatory protein, interleukin 1 beta, transforming growth factor beta 3, and thymosin beta 10) were shown to be differentially expressed by using quantitative real time RT-PCR. Our study shows that RNA amplification is useful for transcriptional profiling with limiting porcine embryonic RNA, and that this novel targeted array can detect differential gene expression during trophoblastic elongation. Finally, our results contribute to an increased understanding of the temporal patterns of expression of known genes controlling conceptus development, as well as identify novel genes also differentially regulated during implantation.

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PubMed2005

Characterization of pig vasa homolog gene and specific expression in germ cell lineage.

The vasa gene is known to be an important factor for germ cell development in both invertebrates and vertebrates. In the present study, we cloned the porcine vasa homolog (Pvh, 2,172 bps) and investigated its expression at mRNA and protein levels. The isolated cDNA had deduced 724 amino acid residues with significant homology to mouse (85%) and human (91%) vasa. In adult tissues, Pvh transcript was restricted to the ovary and testis, and was undetectable in somatic tissues. During preimplantation embryo development, Pvh was transcribed in oocytes and fertilized 2-cell embryos, but not in other preimplantation embryos. In fetal stage, the transcript of Pvh gene was expressed in all fetal stage, except in day 17-18. Immunohistochemical analysis of fetal and adult gonad revealed that the Pvh protein was localized in oocytes and spermatocytes, consistent with mRNA expression. Interestingly, Pvh protein was also observed in proliferating primordial germ cells (PGCs) and freshly isolated PGCs, but not in embryonic germ cells. Our results suggest that Pvh gene can be a useful marker for germ cell development in pigs.

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PubMed2005

Embryotropic effect of insulin-like growth factor (IGF)-I and its receptor on development of porcine preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer.

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine/paracrine growth/survival factor for mammalian embryo development. The present study investigated the temporal expression and regulation of porcine IGF-I receptor (IGF-IR) mRNA and the role of IGF-I on development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. As assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), the level of IGF-IR mRNA expression was high in unfertilized oocytes, 2-cell and 4-cell embryos and gradually decreased in 8-cell embryos, morulae, and blastocysts in both IVF and SCNT series. The IVF or SCNT embryos were cultured with 0, 1, 10, 50, or 100 ng/ml IGF-I for 168 hr. Supplementing with 50 ng/ml IGF-I increased blastocyst formation and the number of cells in inner cell masses (ICMs) in both IVF and SCNT embryos. In a second experiment, more blastocysts were obtained when IVF or SCNT embryos were cultured for the first 48 hr or for the entire 168 hr with 50 ng/ml IGF-I compared to culturing without IGF-I for 48 hr or with IGF-I for the last 120 hr or without IGF-I for the entire 168 hr. Treating IVF or SCNT embryos with 50 ng/ml IGF-I significantly up-regulated IGF-IR mRNA compared to untreated control embryos. In conclusion, the present study demonstrated that IGF-IR mRNA is expressed in porcine IVF and SCNT embryos, and that IGF-I improved the developmental competence of IVF and SCNT embryos through its specific receptors.

Animals↗

PubMed2005

Successful surgical correction of anal atresia in a transgenic cloned piglet.

Inbred strains of pig become indispensable for a wide range of biological studies. In biomedical science, it is generally accepted that somatic cell nuclear transfer (SCNT) technology with inbreed strain of pig is essential for xenotransplantation. In this study, we observed the anal atresia in a cloned pig which was derived from fetal fibroblast of inbreed miniature pig. A presumptive anal site of the cloned pig was excised and the rectum was sutured to apposed skin for treatment. This cloned piglet seemed to be normal with healthy status after surgery. This report can be useful for the treatment of anal atresia of cloned piglets.

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PubMed2004

Role of messenger RNA expression of platelet activating factor and its receptor in porcine in vitro-fertilized and cloned embryo development.

Platelet activating factor (PAF) is known as an autocrine growth/survival factor in mammalian preimplantation embryos. This study investigated the expression of porcine PAF receptor (PAFr) mRNA and its role in porcine in vitro fertilized (IVF) or somatic cell nuclear transfer (SCNT) embryo development. The expression of PAFr mRNA in IVF or SCNT blastocysts was shown by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. Semiquantitative RT-PCR and Southern blot analysis demonstrated that PAFr mRNA was expressed during preimplantation embryo development, it was highly expressed through the 2-cell to 8-cell embryo stage, and it decreased at the morula stage. PAFr mRNA expression was detected steadily in IVF embryos, whereas it was varied at the 2-cell, 4-cell, and blastocyst stages in SCNT embryos. To determine the role of PAF in IVF and SCNT embryo development, embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with different concentrations of PAF (0, 0.037, 0.37, 3.72, or 37.2 nM). The PAF supplement significantly increased the rate of blastocyst formation in SCNT embryos, but not in IVF embryos. The PAF supplement for the entire 168 h of culture showed significantly higher blastocyst formation in SCNT embryos. Upregulation of PAFr mRNA by PAF in SCNT embryos indicated that the embryotrophic effect of PAF was mediated through its functional receptors in SCNT embryos. In conclusion, the present study demonstrated that PAFr mRNA was expressed in porcine IVF and SCNT embryos, and that PAF supplement improved the developmental competence of SCNT embryos through its specific receptors.

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PubMedDate not supplied

Hemichorea-hemiballism in primary diabetic patients: MR correlation.

PURPOSE: The purpose of this work was to describe the characteristic imaging findings and clinical presentations in patients with hemichorea-hemiballism (HC-HB) associated with nonketotic hyperglycemia (NKH) in primary diabetes mellitus (DM). METHOD: The MR findings from six patients with HC-HB associated with NKH in primary DM were evaluated. Their ages ranged from 43 to 81 years. CT was performed on three patients, one of whom underwent a SPECT exam and another who had follow-up MRI. RESULTS: A high-signal putaminal lesion was evident on the T1-weighted images in all cases without edema or mass effect. Three of the six cases also showed high-signal intensities in the caudate. Two cases revealed high-signal intensities in the globus pallidus, and the lesions extended to the midbrain in one patient. The T2-weighted and FLAIR images were more variable. One diffusion-weighted image showed increased signal intensity. All three patients who had postcontrast MRI showed no enhancement. Two of the three patients who had CT studies showed high attenuation and the other isodensity. The SPECT study showed decreased perfusion. In all our patients, the chorea resolved within days to weeks after correction of the underlying hyperglycemia. CONCLUSION: In patients with HC-HB with NKH in primary DM, T1-weighted MR images showed hyperintense lesions of the putamen or caudate. Early recognition of these imaging characteristics may facilitate the diagnosis of primary DM with hyperglycemia and lead to prompt and appropriate therapy.

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