• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.86-92
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• 2019

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.718-724
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• 2007

Considerable attention has been focused on the cloning of mammalian embryos, as a consequence of poor development, in order to enhance the application of genetic engineering. Experiments were conducted to compare the developmental competence of parthenotes and reconstructed (NT) rabbit eggs with fetal fibroblasts (FFs) following various activation regimens. Oocytes and NT eggs were exposed to: electric stimulation (EST, Group 1) and EST followed by 6-dimethylaminopurine (DMAP, Group 2), cycloheximide (CHX, Group 3) or DMAP/CHX (Group 4). Pronuclear (PN) status, cleavage, blastocyst development and the ploidy were assessed. In parthenote groups 1, 2, 3 and 4, the PN formation differed significantly. And, the cleavage and blastocyst rates were 41.7 and 5%, 75.6 and 53.7%, 68 and 36%, 82.1 and 52.6%, respectively, among treatments. Polyploidy was observed in 17.2% of EST plus DMAP and 44.9% of EST plus DMAP/CHX groups. In SCNT groups (Group 1, 2, 3 and 4), the cleavage and blastocyst rates were 28.6 and 7.1%, 58.3 and 29.2%, 56.8 and 24.1%, 64.5 and 27.8%, respectively. The chromosomal composition differed significantly (p<0.05) among treatments. In Group 2 and 3, 53.8% and 81.8% of embryos revealed diploid chromosomal sets, respectively. However, in Group 4, 53.3% of embryos showed abnormal ploidy (mixoploid). Although DMAP or combination with DMAP/CHX resulted in higher in vitro development of rabbit SCNT embryos, higher incidence of chromosomal abnormality may induce problems related to fetal loss of at late stage of development.

• Environmental Analysis Health and Toxicology
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• v.19 no.2
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• pp.191-200
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• 2004

Zinc (Zn) is an essential element in biological process, however inadequate Zn status in general population have been recognized. To update the knowledge for Zn-cadmium (Cd) interaction, we studied the intestinal uptake and transport, and the expression of metal transporter proteins (divalent metal transporter 1, DMT1 ; metal transporter protein 1, MTP1 ; zinc transporter 1, ZnTl ; metallothionein 1 , MT1) in duodenum after Cd exposure using Zn deficient animal model. Rats were led Zn deficient (ZnD, 0.5-1.0 mgZn/kg) or Zn supplemented (ZnS, 50mg Zn/kg) diet for 4 weeks, and followed single administration of $^{109}$ CdCl$_2$orally. The body Zn flatus and tissue Cd concentration were determined at 24 hrs after Cd administration. Total body burden of Cd and Cd absorption index (AI, %) were estimated based on the tissue Cd analyzed. DMT1, MTP1, ZnTl and MT1 mRNA were analyzed by using RT-PCR method. Feeding of Zn deficient diet for 4 weeks produced a reduced body weight gain and a depletion of body Zn. Tissue Cd concentration, body burden of Cd and Cd absorption index were higher in the ZnD diet fed rats than the ZnS diet red rats. Especially, Cd concentration in the small intestine (duodenum, jejunum and ileum) and the colon of FeD diet fed rats were higher markedly than in the FeS diet group. The expression levels of DMT1, MTP1 and ZnT1 mRNA in FeD diet fed rats were similar to the FeS diet. The level of MT1 mRNA expression was significantly lower in the FeD than the FeS diet fed rats. Taken together, theses results indicate that Zn deficiency in diet induce an increased intestinal absorption and tissue retention of Cd, and down -regulate the MT1 expression in the intestine which might be play a part of role in Cd absorption and transport in mammalian. These findings suggest that deficiency of essential metal could be enhanced the toxicity of toxic, non-esstial metals through the metal-metal interaction.

• Development and Reproduction
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• v.7 no.2
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• pp.61-68
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• 2003

Zygote genome should entail a complex process of epigenetic reprogramming including a global DNA demethylation to reach a totipotency or pluripotency during early mammalian development. In this study, we have analyzed methylation patterns in cloned bovine embryos to monitor the epigenetic reprogramming process of donor genomic DNA. Aberrant DNA methylation patterns were observed in various genomic regions of cloned embryos except single-copy gene sequences. The overall genomic methylation status of cloned embryos was quite different from that of normal embryos produced in viかo or in vivo. Abnormal methylation profiles were also specifically represented in trophectoderm cells of cloned embryos, which probably result in widespread gene dysregulation in extraembryonic region or placental dysfunction familiar to cloned animals. Our findings suggest that developmental failures of cloned embryos are due to incomplete epigenetic reprogramming of donor genomic DNA. Understanding the epigenetic reprogramming processes of donor genome will clearly define the faulty development of cloned embryos.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.57-62
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• 1999

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• Proceedings of the Botanical Society of Korea Conference
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• 1985.08b
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• pp.7-18
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• 1985

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• Molecules and Cells
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• v.38 no.11
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• pp.925-935
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• 2015

DNA methylation is a well-characterized epigenetic modification that plays central roles in mammalian development, genomic imprinting, X-chromosome inactivation and silencing of retrotransposon elements. Aberrant DNA methylation pattern is a characteristic feature of cancers and associated with abnormal expression of oncogenes, tumor suppressor genes or repair genes. Ten-eleven-translocation (TET) proteins are recently characterized dioxygenases that catalyze progressive oxidation of 5-methylcytosine to produce 5-hydroxymethylcytosine and further oxidized derivatives. These oxidized methylcytosines not only potentiate DNA demethylation but also behave as independent epigenetic modifications per se. The expression or activity of TET proteins and DNA hydroxymethylation are highly dysregulated in a wide range of cancers including hematologic and non-hematologic malignancies, and accumulating evidence points TET proteins as a novel tumor suppressor in cancers. Here we review DNA demethylation-dependent and -independent functions of TET proteins. We also describe diverse TET loss-of-function mutations that are recurrently found in myeloid and lymphoid malignancies and their potential roles in hematopoietic transformation. We discuss consequences of the deficiency of individual Tet genes and potential compensation between different Tet members in mice. Possible mechanisms underlying facilitated oncogenic transformation of TET-deficient hematopoietic cells are also described. Lastly, we address non-mutational mechanisms that lead to suppression or inactivation of TET proteins in cancers. Strategies to restore normal 5mC oxidation status in cancers by targeting TET proteins may provide new avenues to expedite the development of promising anti-cancer agents.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.39-46
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• 2016

Cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig embryos, because of their greater susceptibility to cryoinjuries, have shown a reduced developmental competence. The aim of this study was to evaluate the survival status of vitrified-warmed porcine embryos. Forced blastocoele collapse (FBC) and non-FBC blastocysts are vitrified and concomitantly cultured in culture media which were supplemented with/without fetal bovine serum (FBS). Porcine vitrified-warmed embryos were examined in four different methods: group A, non-FBC without FBS; group B, non-FBC with FBS; group C, FBC without FBS; group D, FBC with FBS. After culture, differences in survival rates of blastocysts derived from vitrified-warmed porcine embryos were found in group A~D (39.5 (A) vs 52.5 (B) and 54.8 (C) vs 66.7% (D), respectively, p<0.05). Reactive oxygen species (ROS) level of survived blastocysts was lower in group D than that of another groups (p<0.05). Moreover, total cell number of survived blastocysts was higher in group D than that of other groups (p<0.05). Otherwise, group D showed significantly lower number of apoptotic cells than other groups ($2.0{\pm}1.5$ vs $3.2{\pm}2.1$, $2.8{\pm}1.9$, and $2.7{\pm}1.6$, respectively, p<0.05). Taken together, these results showed that FBS/FBC improves the developmental competence of vitrified porcine embryos by modulating intracellular levels of ROS and the apoptotic index during the vitrification/warming procedure. Therefore, we suggest that FBS and FBC are effective treatment techniques during the vitrification/warming procedures of porcine blastocysts.

• Journal of Korean Neurosurgical Society
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• v.63 no.5
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• pp.566-578
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• 2020

Objective : Radiation is known to induce autophagy in malignant glioma cells whether it is cytocidal or cytoprotective. Dexamethasone is frequently used to reduce tumor-associated brain edema, especially during radiation therapy. The purpose of the study was to determine whether and how dexamethasone affects autophagy in irradiated malignant glioma cells and to identify possible intervening molecular pathways. Methods : We prepared p53 mutant U373 and LN229 glioma cell lines, which varied by phosphatase and tensin homolog (PTEN) mutational status and were used to make U373 stable transfected cells expressing GFP-LC3 protein. After performing cell survival assay after irradiation, the IC₅₀ radiation dose was determined. Dexamethasone dose (10 μM) was determined from the literature and added to the glioma cells 24 hours before the irradiation. The effect of adding dexamethasone was evaluated by cell survival assay or clonogenic assay and cell cycle analysis. Measurement of autophagy was visualized by western blot of LC3-I/LC3-II and quantified by the GFP-LC3 punctuated pattern under fluorescence microscopy and acridine orange staining for acidic vesicle organelles by flow cytometry. Results : Dexamethasone increased cell survival in both U373 and LN229 cells after irradiation. It interfered with autophagy after irradiation differently depending on the PTEN mutational status : the autophagy decreased in U373 (PTEN-mutated) cells but increased in LN229 (PTEN wild-type) cells. Inhibition of protein kinase B (AKT) phosphorylation after irradiation by LY294002 reversed the dexamethasone-induced decrease of autophagy and cell death in U373 cells but provoked no effect on both autophagy and cell survival in LN229 cells. After ATG5 knockdown, radiation-induced autophagy decreased and the effect of dexamethasone also diminished in both cell lines. The diminished autophagy resulted in a partial reversal of dexamethasone protection from cell death after irradiation in U373 cells; however, no significant change was observed in surviving fraction LN229 cells. Conclusion : Dexamethasone increased cell survival in p53 mutated malignant glioma cells and increased autophagy in PTEN-mutant malignant glioma cell but not in PTEN-wildtype cell. The difference of autophagy response could be mediated though the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling pathway.

• Environmental Analysis Health and Toxicology
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• v.19 no.4
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• pp.359-366
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• 2004

Iron (Fe) is an essential metal in biological processes, which maintains a homeostasis in the human body. Divalent metal transporter 1 (DMT1) has been known as an iron transporting membrane protein, which is involved in the uptake Fe at the apical portion of intestinal epithelium, and may transport Fe across the membrane of acidified endosome in peripheral tissues. In this study, we studied the tissue distribution of DMT1 in the Fe supplemented (FeS) diet fed rats, and the regulation of DMT1 expression by depleting body Fe. Sprague-Dawley rats were divided into two groups, and fed FeS (120 mg Fe/kg) diet or Fe deficient (FeD, 2∼6 mg Fe/kg) diet for 4 weeks. The evaluation of body Fe status was monitored by measuring sFe, UIBC and tissue Fe concentration. Additionally, DMT1 mRNA levels were analyzed in the peripheral tissues by using the quantitative real time RT-PCR method. In the FeS diet fed rats, the tissue Fe was maintained at a relatively high level, and DMT1 was eventually expressed in all tissues studied. DMT1 was highly expressed in the testis, kidney and spleen, while a moderate levels of DMT1 expression was detected in the brain, liver and heart. In the digestive system, the highest level of DMT1 was found in the duodenum. Feeding the FeD diet caused a reduced body weight gain and depletion of body Fe with finding of decreased sFe, increased UIBC and decreased tissue Fe concentration. The depletion of body Fe upregulated DMT1 expression in the peripheral tissue. The expression of DMT1 was very sensitive to the body Fe depletion in the small intestine, especially in the duodenum, showing dramatically higher levels in the FeD rats than those of the FeS group. In the FeD diet fed animals, the expression of DMT1 was low significantly in other tissues compared with the duodenum. The expression of DMT1, however, was 60∼120% higher in the testis, kidney and spleen, and 30∼50% higher in the lung, liver and heart, compared to the FeS diet fed rats. In summary, DMT1 expression was ubiquitous in mammalian tissue, and the level of expression was the organ-dependent. The expression of DMT1 in peripheral tissues was upregulated by depletion of body Fe. Duodenum was the most sensitive tissue among organs studied during Fe depletion, and expressed the greatest level of DMT1, while other tissues were less higher than in duodenum. This study supports that DMT1 plays a role in maintaining the body Fe level through intestinal uptake as well as homeostasis of Fe in the peripheral tissue.