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ABSTRACT: A worldwide flu pandemic occurred in 2009, resulting in many victims and high social damages. In the A (H1N1) virus spreading process, the pig is the intermediate host, and this virus is amplified and genetically changed through recombination in pigs. The objective of this study was to develop influenza-resistant pigs. In interferon-α and interferon-γ treated cells, the porcine Mx2 protein has been observed near the nuclear envelope, which consequently has been linked with inhibition of influenza virus proliferation. Therefore, we attempted to produce transgenic (TG) pigs overexpressing the Mx2 gene by somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were transfected with the cytomegalovirus vector, which includes the porcine Mx2 gene. The established transgenic cell was injected into the enucleated ooplasm to produce Mx2-TG cloned embryos. In total, 511 female TG porcine embryos were transferred to 5 surrogates. Two recipients were diagnosed pregnant (pregnancy rate, 40%) on Day 25. On Day 114, 6 fetuses and 4 mummies were collected. The PCR analysis concluded that there was no integration of the Mx2 gene. Then, a male Mx2-TG cell line was established to use as donor cell of SCNT. In total, 547 male TG-SCNT embryos were produced. Of these, 38 embryos were cultured in vitro to confirm the developmental capacity of the embryos. Among these porcine SCNT-TG embryos, 26 embryos (68.4%) cleaved and 5 (13.2%) developed to the blastocyst stage. The PCR analysis confirmed that all male TG-SCNT blastocysts were for integration of the Mx2 gene. The remaining 509 male embryos were transferred to 5 surrogates. Two recipients (pregnancy rate, 40%) were diagnosed pregnant at Day 25. To date, 1 of the surrogate has maintained pregnancy and another recipient gave birth to 9 piglets. Two days after birth, 2 piglets died and the remaining piglets remain healthy. Verification analysis of gene targeting and resistance to influenza is in progress. This study has presented new possibilities of production of influenza virus resistant pig by SCNT for translational research.
Reproduction Fertility and Development 12/2012; 25(1):309. · 2.11 Impact Factor
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ABSTRACT: Endocrine-disrupting chemicals (EDC) can bind to the hormone receptor and induce an unexpected hormone response to activate oestrogen receptor (ER)- and androgen receptor (AR)-mediated signalling pathways. Among EDC, bisphenol A (BPA) has a detrimental effect on the endocrine system and is suspected to promote human breast and ovarian cancers. Recent studies have reported that phthalate can disrupt the endocrine system and has a weak estrogenic activity with binding to ER. In this study, we demonstrated whether BPA and dibutyl phthalate (DBP) stimulate the proliferation of prostate cancer cells, LNCaP cells, which have both ER and AR. We evaluated the proliferative rate of LNCaP cells following BPA and DBP treatment using a cell viability assay compared with EtOH treatment as a negative control. Further, we examined the alteration of cell cycle-related gene expressions and TGF-β signalling molecules by semiquantitative RT-PCR. Both BPA and DBP increased LNCaP cell growth more than 2-fold. Moreover, these EDC altered transcriptional expressions of cell cycle-related genes, cyclin D1 and p21, at 6h in LNCaP cells after exposure of BPA and DBP. Like 17β-oestradiol (E2) and dihydrotestosterone (DHP), treatments of BPA and DBP lead to an increase of the transcriptional levels of c-myc and c-fos in LNCaP cells from 30min to 6h. In addition, BPA and DBP decreased the protein level of not only p-smad but also total smad, suggesting that these EDC can affect the molecules of the TGF-β signalling pathway. It was of interest that these effects of EDC were reversed by an antagonist of ER or AR signalling pathways in these prostate cancer cells. These results suggest that BPA and phthalate can alter various gene expressions in TGF-β signalling molecules and stimulate cell growth in prostate cancer cells in vitro. In addition, the growth of prostate cancer cells was stimulated following the exposure of E2, DHT, and DBP in vivo. Taken together, these results indicate the potential of BPA and phthalate in the carcinogenesis of prostate cancer by the oestrogen or androgen-dependent signalling pathway.
Reproduction Fertility and Development 12/2012; 25(1):245-6. · 2.11 Impact Factor
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ABSTRACT: Despite recent efforts to improve culture systems, the developmental competence of in vitro-matured (IVM) porcine oocytes is still inferior compared with those that have been in vivo matured. In pigs, cumulus-oocyte complexes (COC) are usually aspirated from 3- to 7-mm follicles and matured for 42 to 44h in vitro. In this study, we compared oocytes obtained from large-sized (≥8-mm) and medium-sized (3- to 7-mm) follicles in terms of nuclear maturation, intracellular reduced glutathione levels, gene expression, and embryo development after IVM. In the control group, COC (n=521) were aspirated from 3- to 7-mm follicles and matured for 22h with hormones (eCG/hCG) and subsequently matured in vitro for 20 to 22h without hormones at 39°C, 5% CO(2). In the large follicle (LF) group, COC (n=256) were obtained from follicles larger than 7mm and were subjected to IVM reduced for 18h. The maturation medium was TCM-199 supplemented with 0.6mM cysteine, 0.91mM sodium pyruvate, 10ngmL(-1) of epidermal growth factor, 75µgmL(-1) of kanamycin, 1µgmL(-1) of insulin, and 10% (vol/vol) porcine follicular fluid without hormones. Nuclear status and reduced glutathione content in oocytes were investigated by Hoechst 33342 staining and CellTracker Blue (CMF2HC; Invitrogen, Carlsbad, CA, USA), respectively. The abundance of messenger RNA of genes reflecting the developmental competence of oocytes was analysed in cumulus cells by real-time PCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as the reference. In addition, oocytes were subjected to parthenogenetic activation to assess in vitro embryo developmental competence. Data were analysed by Student's t-test using SPSS version 17.0 (IBM Corporation, Armonk, NY, USA) and presented as means. All experiments were replicated at least three times. The average frequency of ovaries having ≥8-mm follicles was 2.3% (44/1953 in 11 replicates). Before IVM, the nuclear-stage oocytes from ≥8-mm follicles were as follows: germinal vesicle stage=15.2%; metaphase I (MI) stage=55.4%; anaphase I and telophase I (AI+TI) stages=15.8%; and metaphase II (MII) stage=13.6%. After 6h of IVM, 4.2% of oocytes were at the germinal vesicle stage and frequencies of the MI, AI+TI, and MII stages were 43.6, 9.4, and 42.8%, respectively. After 18h, IVM frequencies of the MI and MII stages were 13.0 and 87.0%, respectively. Oocytes of the LF group showed a significant (P<0.001) increase in intracellular reduced glutathione level (1.41 v. 1.00) compared with the control (42- to 44-h matured oocytes). Cumulus cells in the LF group showed lower (P<0.1) messenger RNA expression of COX-2 (cyclooxygenase-2) and TNFAIP6 (tumor necrosis factor, α-induced protein 6), and higher (P<0.1) expression of PCNA (proliferating cell nuclear antigen) and Nrf2 (NF-E2-related factor 2) compared with the control. After parthenogenetic activation, the oocytes from the LF group had significantly (P<0.05) higher blastocyst rates and total cell numbers in blastocysts than did the control group (90.1% and 73.6 v. 50.5% and 55.3, respectively). In conclusion, oocytes from preovulatory LF require only 18h to complete maturation in vitro, and their developmental competence is higher than those obtained from MF. Although limited, oocytes from ≥8-mm follicles offer an alternative source of material for the production of transgenic pigs by somatic cell nuclear transfer.
Reproduction Fertility and Development 12/2012; 25(1):281. · 2.11 Impact Factor
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ABSTRACT: The establishment of porcine embryonic stem cells (pESC) using in vitro-produced blastocysts derived from IVF, parthenogenesis (PA), and somatic cell nuclear transfer (SCNT) have a great potential. However, porcine in vitro-produced blastocysts show a lower quality than do in vivo-derived blastocysts. In this study, to improve in vitro-derived blastocyst quality, and then to establish pESC, we treated IVF embryos and PA embryos with resveratrol (RES), porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), and β-mercaptoethanol (β-ME). The control system was produced using M199 medium in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The modified group was produced using M199 with 2µM RES in IVM and PZM5 with 10ngmL(-1) pGM-CSF, 2µM RES, and 10µM β-ME in IVC. Data were analysed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA) using Duncan's multiple range test. In total, 1210 PA, 612 IVF, and 5 SCNT embryos were evaluated (for SCNT, we examined only the control system). We observed that overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (P<0.05) in the modified system (54.5%) compared with the control system (43.4%) in PA and hatched blastocysts rates at Day 6 and 7 were also increased significantly. Total blastocyst cell numbers were significantly higher (P<0.05) in the modified system (55.1) compared with the control system (45.6). Hatching rate of Day 7 IVF blastocysts was also significantly increased. After seeding porcine blastocysts, the attachment rates were higher in the modified system (32.2% in PA and 36.2% in IVF; not examined in SCNT) than the control system (19.5% in PA, 26.6% in IVF, and 40.0% in SCNT). In addition, colonization rates and cell line derivation rates were higher in the modified system than in the control system. Colonization rates of modified system were 13.1% for PA and 17.75% for IVF embryos, whereas the control system generated 2.4% for PA, 10.8% for IVF, and 20.0% for SCNT. We also investigated the correlation between blastocyst state and attachment rate. The highest attachment rate is in hatched blastocyst (78.35±15.74%). Therefore, the modified system increased quality of porcine blastocysts in vitro produced, and subsequently increased attachment rates. The cell line derivation rates were 2.4% (PA), 4.2% (IVF), and 20.0% (SCNT) in control group. In modified system, they were 7.2% (PA) and 10.0% (IVF). We established 3 cell lines from PA blastocysts (1 cell line in control system and 2 cell lines in modified system) and 1 cell line from SCNT-control system. All cell lines showed alkaline phosphatase activity and expressed pluripotent markers. In conclusion, the modified system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of in vitro-produced porcine blastocysts, and subsequently increased derivation rates of putative pESC.
Reproduction Fertility and Development 12/2012; 25(1):299. · 2.11 Impact Factor
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ABSTRACT: Previous studies have demonstrated that treatment of cloned embryos with trichostatin A (TSA) or scriptaid, inhibitors of class I and II histone deacetylases (HDAC), significantly enhanced their developmental competence. In the present study, we investigated the effects of sirtinol, an inhibitor of class III HDAC, on the embryonic development of porcine cloned embryos. Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) using Duncan's multiple range test and all experiments were replicated at least 5 times. In experiment 1, 648 parthenotes were divided into 4 groups (0-, 6-, 12-, and 24-h sirtinol treatment after activation) to investigate optimal treatment time using 100µM sirtinol. The cleavage rate of the 24-h treatment group (81.3%) was significantly (P<0.05) decreased compared with the 12-h treatment group (88.4%) but there was no difference compared with the control (86.9%) and 6-h treatment groups (86.9%). The parthenotes treated with sirtinol for 12h after activation had a significantly higher blastocyst formation rate and total cell number in blastocysts (50.5% and 66.9, respectively) than the control (39.4% and 54.1, respectively). In experiment 2, 806 cloned embryos were divided into 5 groups (0, 50, 100, 150, and 200µM sirtinol treatment for 12h after activation) to investigate optimal concentration. There was no significant difference in cleavage rate. The rate of blastocyst formation and total cell number in blastocysts were significantly (P<0.05) improved by treatment with 150µM sirtinol for 12h after activation (28.8% and 51.0, respectively) compared with the control (17.5% and 37.1, respectively). The total cell number in blastocysts was also significantly increased in 50 and 200µM groups (47.9 and 48.4, respectively) compared with the control (37.1). In experiment 3, we examined the effects of 150µM sirtinol treatment for 12h after activation with or without 5nM TSA on in vitro embryonic development after somatic cell nuclear transfer. The rate of blastocyst formation was significantly improved in sirtinol-treated and TSA-treated groups (30.9 and 31.3%, respectively) but not in the sirtinol with TSA group (27.6%) compared with the control (21.7%). The total cell number in blastocysts was significantly increased by treatment with sirtinol and TSA together (73.9) compared with the control (49.0) but there was no difference in only sirtinol- (59.8) or TSA- (59.2) treated groups. There was no significant difference in cleavage rate among groups. Our results suggest that sirtinol improves the embryonic development of porcine cloned embryos and sirtinol with TSA synergistically increases the blastocyst quality.
Reproduction Fertility and Development 12/2012; 25(1):166. · 2.11 Impact Factor
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ABSTRACT: As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.
Cancer gene therapy 06/2012; 19(8):517-22. · 3.13 Impact Factor
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ABSTRACT: This study investigated the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the developmental potential of porcine in vitro-fertilized (IVF) embryos in chemically and semidefined (with BSA) medium. In experiment 1, zygotes were treated with different concentrations of pGM-CSF (0, 2, 10, 100 ng/mL). The results indicated that 10 ng/mL pGM-CSF significantly (P < 0.05) increased blastocyst development and total cell number (15.1% and 53.5, respectively) compared with the control (6.1%, and 38.8, respectively). Comparing blastocyst formation, early and expanded blastocyst formation was significantly higher in the 10 ng/mL-pGM-CSF group than in the control on Days 6 and 7 of the culture period. However, there was no significant difference in cleavage rate. Experiment 2 demonstrated that pGM-CSF influenced the percentage of blastocyst formation and total cell number when pGM-CSF was added during Days 4 to 7 (14.6% and 53.9, respectively) or Days 0 to 7 (15.2% and 54.0, respectively) compared with the control (7.8% and 43.1, respectively) and compared with Days 0 to 3 (8.7% and 42.5, respectively). Similarly, early blastocyst formation rates were significantly higher at Days 4 to 7 than in the control, and expanded blastocyst formation was significantly higher at Days 4 to 7 or Days 0 to 7. No significant difference in cleavage rates appeared among the groups. In experiment 3, in the presence of BSA, pGM-CSF also increased the percentage of embryos that developed to the blastocyst stage and the total cell number (20.3% and 59.8, respectively) compared with the control (14.9% and 51.4, respectively), whereas there was no significant difference in cleavage rate. Experiment 4 found that the total cell number and the number of cells in the inner cell mass (ICM) were significantly increased compared with the control when zygotes were cultured in either porcine zygotic medium (PZM)-3 or PZM-4 supplemented with 10 ng/mL pGM-CSF. The number of trophectoderm (TE) cells was significantly higher in PZM-3 medium supplemented with pGM-CSF than in the control, and the number tended to increase (P = 0.058) in PZM-4 medium supplemented with pGM-CSF. The ratio of inner cell mass to trophectoderm cells was significantly higher in PZM-4 supplemented with 10 ng/mL pGM-CSF, but not in PZM-3. In experiment 5, it was found that the male pronuclear formation rate, monospermic penetration and sperm/oocyte were 95.4%, 37.2%, and 2.4, respectively. Together, these results suggest that pGM-CSF may have a physiological role in promoting the development of porcine preimplantation embryos and regulating cell viability and that addition of pGM-CSF to IVC medium at Days 4 to 7 or 0 to 7 improves the developmental potential of porcine IVF embryos.
Theriogenology 12/2011; 77(6):1186-97. · 1.96 Impact Factor
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ABSTRACT: Preeclampsia is a pregnancy-specific disease characterised by de novo development of concurrent hypertension and oxidative stress in the placenta. The human placenta is a highly vascularized organ whose major function is to allow maternal-fetal exchange of solutes such as Ca(2+) and oxygen. The transient receptor potential cation channel subfamily is known to contain channels activated by such various stimuli as intracellular Ca(2+) membrane potential, cold and pH. However, signalling mechanisms mediating hormonal regulation of Mg(2+) inorganic phosphate channels during the placenta duration of pregnancy are incompletely understood. We examined the expression of cell membrane Mg(2+) inorganic channels in 3 sections (fetal-, central-, maternal-) of preeclamptic placenta (PEP) and from placental cell lines, BeWo, JEG3 and hPC (isolated during the first trimester) by real-time PCR and Western blot analysis. Placental tissues (~3cm) from women (n=75) between the ages of 28 to 45 years undergoing normal or Caesarean delivery were dissected into 1-cm sections. Data were analysed by one-way ANOVA, followed by Tukey's multiple comparison test. P<0.05 was considered to be statistically significant. During preterm labour, human placental expression of Mg(2+) inorganic channel mRNA and protein fluctuated in the 3 sections of PEP compared with normal placenta. In hPC, the expression of Mg(2+) inorganic channel (TRPM6/7, MagT1, NIPA2, SLC41A1 to 3 and SLC34A1 to 3) mRNA and protein were decreased in placental cell lines by hypoxic stress (2% O(2)/93% N(2)/5% CO(2)) compared with normoxia (20% O(2)/75% N(2)/5% CO(2)). The levels of Mg(2+) inorganic channel mRNA and protein were distinctly expressed between BeWo and JEG3 cells. These results indicate that placental Mg(2+) inorganic channels play potential roles in differential sections of placenta between normal and PEP, suggesting that induced Mg(2+) inorganic channels of PEP may be involved in preeclamptic stress in human placenta and placental cell lines, which are a determinant factor affecting calcium transfer.
Reproduction Fertility and Development 12/2011; 24(1):153. · 2.11 Impact Factor
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ABSTRACT: The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in matured oocytes and subsequent embryonic development after parthenogenetic activation (PA) and IVF. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. In experiment 1, a total of 1146 cumulus-oocyte complexes (COC) were divided into 5 groups (0, 0.1, 0.5, 2.0 and 10.0μM resveratrol). In the nuclear maturation after 44-h IVM, the groups of 0.1, 0.5 and 2.0μM (83.0, 84.1 and 88.3%, respectively) had no significant difference compared to the control group (84.1%). The group of 10.0μM decreased the nuclear maturation (75.0%) significantly (P<0.05). In experiment 2, a total of 300 matured oocytes were examined for the effects of different resveratrol concentrations (0, 0.5, 2.0 and 10.0μM) on porcine oocyte intracellular GSH and ROS levels. The groups of 0.5 and 2.0μM showed a significant (P<0.05) increase in intracellular GSH levels (1.3 and 1.3, respectively) compared with the control and 10.0μM groups (1.0 and 1.0, respectively). The intracellular ROS level of oocytes matured with 2.0μM resveratrol (0.4) was significantly (P<0.05) decreased compared to other groups (control: 1.0; 0.5μM: 0.6; and 10.0μM: 0.7). In experiment 3, lower expression of apoptosis-related genes (Bax, Caspase-3 and Bak) was observed in matured oocytes treated with 2.0μM resveratrol when compared with that of the control (P<0.05). In experiment 4, a total of 728 oocytes were divided into 4 groups (control, 0.5, 2.0 and 10.0μM) and examined subsequent to embryonic development after PA. Oocytes treated with 2.0μM resveratrol during IVM had a significantly higher cleavage (CL) rate, blastocyst (BL) formation rate and total cell numbers (TCN) after PA compared with those of the control (2.0μM: 96.6%, 62.1% and 49.1 vs control: 88.3%, 48.8% and 41.4, respectively) and the 10.0μM groups (87.3%, 41.4% and 40.9, respectively). Oocytes treated with 0.5μM resveratrol (87.2%, 50.5% and 48.6, respectively) during IVM had significantly higher TCN, but there were no differences in CL and BL formation rates. In experiment 5, a total of 935 oocytes in 3 groups (control, 2.0 and 10.0μM resveratrol) were conducted in IVF. The BL formation rate and TCN were significantly higher in the group of 2.0μM resveratrol (20.5% and 54.0, respectively) than the control (11.0% and 43.4, respectively) and 10.0μM group (11.7% and 45.0, respectively), but there was no significant difference in CL rate. In conclusion, 2.0μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH concentration, decreasing the ROS level and decreasing apoptosis-related gene expression during oocyte maturation.
Reproduction Fertility and Development 12/2011; 24(1):207. · 2.11 Impact Factor
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ABSTRACT: Trans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoids family. Trans-ε-viniferin is isolated from Vitis amurensis, 1 of the most common wild grapes in Korea, Japan and China. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after IVF or parthenogenesis (PA). At the laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Chungbuk National University, trans-ε-viniferin was purified from the leaves and stems of Vitis amurensis. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. First, in total, 594 cumulus-oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0μM) with 10% porcine follicular fluid, 10 IUmL(-1) of eCG and 10 IUmL(-1) of hCG. After 22h in maturation culture, the COC were cultured in hormone-free medium supplemented with various concentrations of trans-ε-viniferin for an additional 22h and then nuclear maturation was evaluated. Second, in total, 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0μM) on porcine oocyte intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. Lastly, the developmental competence of oocytes matured with different concentrations of trans-ε-viniferin (0, 0.5 and 5.0μM) was evaluated after IVF or PA. In total, 711 embryos were evaluated. As results, we observed that trans-ε-viniferin treatment during IVM did not improve the nuclear maturation of oocytes in any group (84.2, 86.6, 85.5, 83.3 and 79.2%, respectively), but significantly increased (P<0.05) intracellular GSH levels in the 0.5μM group (0μM vs 0.5μM; 14.6 vs 16.8 pmoloocyte(-1)) and reduced ROS levels (0μM vs 0.5μM and 50μM; 174.6 vs 25.7 and 23.8 pixeloocyte(-1)). Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after IVF, but total cell numbers were significantly higher (P<0.05) in the 0.5 and 5.0μM treatment groups (53.6±4.0 and 47.9±3.1) compared to the control group (36.4±2.2). The PA embryos showed similar results; there were no significant differences in cleavage rates and blastocyst formation rates, but the total cell number significantly increased in the 0.5 and 5.0μM treatment groups (59.6±4.2 and 60.8±4.6) compared to the control group (43.1±2.1). In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased total cell number of blastocysts, possibly through increasing intracellular GSH synthesis and reducing ROS levels.
Reproduction Fertility and Development 12/2011; 24(1):207-8. · 2.11 Impact Factor
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ABSTRACT: Because endocrine disrupting chemicals may interfere with the endocrine systems of our body and have an oestrogenic activity, we evaluated the effects of bisphenol A (BPA) on the transcriptional levels of altered genes in oestrogen receptor (ER)-positive BG-1 ovarian cancer cells. A microarray and RT-qPCR were employed to detect gene alterations in these cells following treatments. In this study, treatment with 17-β-oestradiol (E2) or BPA increased mRNA levels of E2-responsive genes related to apoptosis, cancer and cell cycle, signal transduction and nucleic acid binding and so on. Parallel with the microarray data, the mRNA levels of some altered genes including RAB31_member RAS oncogene family (U59877), cyclin D1 (X59798), cyclin-dependent kinase 4 (U37022), IGF-binding protein 4 (U20982) and anti-mullerian hormone (NM_000479) were significantly induced by E2 or BPA in this cell model. These results indicate that BPA in parallel with E2 induced the transcriptional levels of E2-responsive genes in an ER-positive BG-1 cells. In conclusion, these microarray and RT-qPCR results indicate that BPA, a potential weak oestrogen, may have an oestrogenic effect by regulating E2-responsive genes in ER-positive BG-1 cells and that BG-1 cells would be the best in vitro model to detect these oestrogenic endocrine disrupting chemicals.
Reproduction Fertility and Development 12/2011; 24(1):188. · 2.11 Impact Factor
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ABSTRACT: The aim of the present study was to investigate whether the effects of vascular endothelial growth factor (VEGF) on porcine cumulus oocyte complexes (COCs) and subsequent blastocyst formation following in vitro fertilization are attributable to improved fertilization and cytoplasmic maturation. Porcine COCs were cultured for 42 h in TCM199 medium with 5 ng/mL human recombinant VEGF, and the resultant metaphase II oocytes were fertilized in vitro. COCs without VEGF supplementation served controls. Supplementation with VEGF during in vitro maturation (IVM) significantly (P < 0.05) improved the blastocyst formation rate and total cell number (46.7 ± 3.1% and 82.8 ± 6.7, respectively) compared with controls (32.5 ± 3.4% and 64.1 ± 5.6, respectively). On day 2, the percentage of four-cell stage embryos was significantly higher in the VEGF-matured group (49.1 ± 2.7%) than in the control (33.1 ± 5.8%), and the percentage of two-cell stage embryos was significantly higher in the control group (10.4 ± 1.4%) than in the VEGF-matured group (6.6 ± 0.9%). At 10 h after the onset of in vitro fertilization (IVF), oocytes with two pronuclei were considered as monospermically or normally fertilized, and oocytes with more than two pronuclei were considered as polyspermically fertilized. Monospermy was significantly higher in VEGF-matured oocytes (47.2 ± 4.3%) than in the control (20.0 ± 2.4%), and polyspermy and sperm penetration per oocyte were significantly higher in the control group (54.4 ± 3.8% and 2.3 ± 0.1, respectively) than in the VEGF-matured oocytes (43.9 ± 3.6% and 1.8 ± 0.1, respectively). Supplementation with VEGF during IVM significantly (P < 0.05) improved male pronuclear formation as compared with the control (91.1 ± 1.9 vs 74.4 ± 3.8%). Type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes (78.0%) than in the control (52.1%). These results suggest that VEGF supplementation during IVM enhanced the developmental potential of porcine IVF embryos through higher male pronuclear formation and higher monospermic fertilization rates as a consequence of improved cytoplasmic maturation.
Theriogenology 07/2011; 76(1):153-60. · 1.96 Impact Factor
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ABSTRACT: Calbindin-D28k (CaBP-28k), a calcium-binding protein, buffers intracellular Ca(2+) and eventually has antiapoptosis properties in neuron, osteoblast, and male germ cells. Although endometrial cancer is the most common invasive gynecological malignancy, CaBP-28k expression in apoptosis signalling is poorly understood. In this study, we investigated whether CaBP-28k expression is regulated by H(2)O(2)-induced apoptosis signalling in human endometrial Ishikawa cells. Ishikawa cells were treated with H(2)O(2) in a dose-dependent manner (0, 0.25, 0.5, 1.0, 1.5, 2mM) and a time-dependent manner (0, 15, 30, 60, 90, 120min). The protein expressions of Bax, p53, and caspase 3 were determined by Western blot analysis. Treatment of Ishikawa cells with H(2)O(2) induced an increase in Bax and p53 expression at the translational level at 1mM for 1h. Interestingly, overexpression of CaBP-28k caused a decrease in Bax, p53, and caspase 3 on H(2)O(2)-induced apoptosis in the Ishikawa cells. These results suggest that expression of CaBP-28k blocked up-regulation of apoptotic gene expression. In addition, knockdown of CaBP-28k expression using a small inhibitory RNA resulted in an elevation of H(2)O(2)-induced cell death and a increase in Bax, p53, and caspase 3 in H(2)O(2)-induced apoptosis, whereas cell survival was increased in overexpressing CaBP-28k cells, providing additional evidence that the induction of CaBP-28k expression may be associated with survival signalling during H(2)O(2)-mediated oxidative cell death. Taken together, these results imply that CaBP-28k expression is involved in the apoptotic pathway and may play a role as an antiapoptotic gene in the human endometrial Ishikawa cells.
Reproduction Fertility and Development 01/2011; 23(1):195. · 2.11 Impact Factor
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ABSTRACT: The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in the female reproductive tract and is one of the regulatory molecules that mediate maternal effects on the growth and development of pre-implantation embryos in several species. The objective of the present study was to investigate the effects of porcine GM-CSF (pGM-CSF) on the developmental potential of porcine IVF embryos. All experiments were performed with zygotes that were produced in vitro and cultured in porcine zygote medium-3-polyvinyl alcohol-based medium. Data were analysed with PASW statistics-17 (SPSS Inc., Chicago, IL) using Duncan's multiple range test. A total 865 zygotes in 4 replicates were used with different concentrations of pGM-CSF (0, 2, 10, 100ngmL(-1)) in Experiment 1. It was demonstrated that 10ngmL(-1) of pGM-CSF could increase (15.1±2.2) blastocyst development significantly (P<0.05) compared with the control (6.1±0.7). There was no effect on cleavage rate. In blastocyst formation, early and expanded blastocysts were significantly (P<0.05) higher in the 10ngmL(-1) of pGM-CSF group compared with the control. In Experiment 2, a total 839 zygotes with at least 5 replicates in each group were used, and whether pGM-CSF would act to increase blastocyst yield before or after Day 4 development was tested. Zygotes were cultured with the following treatments: 1) zygotes cultured with fresh porcine zygote medium-polyvinyl alcohol medium from Days 0 to 7 post-insemination as a control; 2) medium supplemented with 10ngmL(-1) of pGM-CSF from Days 0 to 4 followed by no pGM-CSF from Days 4 to 7; 3) medium alone from Days 0 to 4 followed by supplementation with 10ngmL(-1) of pGM-CSF from Days 4 to 7; and 4) medium supplemented with 10ngmL(-1) of pGM-CSF from Days 0 to 7. As compared with the controls (7.8±0.7), pGM-CSF influenced the percentage of blastocyst formation when pGM-CSF was added from Days 4 to 7 (14.6±1.6) or Days 0 to 7 (15.2±1.8), but not from Days 0 to 4 (8.7±1.5). Similarly, the early blastocyst formation rates were significantly higher in the Day 4 to 7 culture period compared with the control, and expanded blastocyst formation was significantly higher in the Day 4 to 7 and Day 0 to 7 culture periods. There was no significant different in cleavage rate among these groups. In conclusion, these data suggest that supplementation of pGM-CSF in in vitro culture medium at Days 4 to 7 or Days 0 to 7 promotes the developmental potential of porcine IVF embryos.
Reproduction Fertility and Development 01/2011; 23(1):207-208. · 2.11 Impact Factor
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ABSTRACT: The semen storage medium and the embryo culture environment are important for the further development of pre-implantation embryos, and these environments provide an ideal habitat for the propagation of a variety of microorganisms. Even though all precautions are taken to prevent contamination, this happens frequently during IVF and in vitro culture (IVC). Therefore, the aim of the present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen (liquid semen was prepared according the method of (Weitze 1990 Reprod. Dom. Anim., 231-253 suppl. 1), and purchased from the Veterinary Service Laboratory, Department of Livestock Research (Yongin city, Gyeonggi-do, Korea) in our laboratory for IVF experiments because of a lack of fresh semen. Antibiotics were added to the frozen semen extender (kanamycin and gentamicin) and IVC medium (gentamicin) to further inhibit the growth of microorganisms. Nevertheless, microorganisms were observed to proliferate in the IVC drop when culturing IVF embryos using frozen semen. Three samples were randomly taken from the liquid semen, frozen semen, and egg yolk. Contaminated IVC medium, frozen-thawed semen, liquid semen, and egg yolk were cultured in de Man, Rogosa, and Sharpe agar medium. Identical whitish colonies were detected in the contaminated IVC drop, frozen-thawed semen samples, and egg yolk, but no colonies were formed in liquid semen samples. Identical gram-negative and rod-shaped bacteria were found in both the frozen-thawed semen sample and the contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to the manufacturer's instructions, with an identification value of 94.3% and a T index of 0.88. Antibiotic susceptibility tests were done according to CLSI (Wayne, PA) by using an ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole, trimethoprim, norfloxacin, and ciprofloxacin test. Among them, E. cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, and kanamycin but were susceptible to tetracycline, oxytetracycline, sulfamethoxazole and trimethoprim, norfloxacin, and ciprofloxacin. We suggest, based on these findings, that the sources of contamination might be from egg yolk. Therefore, synthetic semen extenders, which are free of egg yolk, might be considered during semen preparation.
Reproduction Fertility and Development 01/2011; 23(1):208. · 2.11 Impact Factor
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ABSTRACT: In the present study, pig cumulus-oocyte complexes were cultured in medium supplemented with different concentrations (0, 5, 50, and 500ngmL(-1)) of vascular endothelial growth factor (VEGF), and then the maturation and intracellular glutathione (GSH) concentration of oocytes were examined. In addition, the development of oocytes matured with different concentrations of VEGF after parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT) was observed. Although the maturation rate of oocytes was not affected by VEGF concentrations (81.13±2.61%, 83.93±1.97%, 82.14±4.03%, 75.24±2.68%, respectively), the intracellular GSH concentrations of oocytes matured with 5 and 50ngmL(-1) VEGF were significantly higher (12.68±0.08, 12.33±0.53pMol/oocyte, respectively) than those of oocytes matured with 0 or 500ngmL(-1) VEGF (10.19±0.66, 10.54±0.54pMol/oocyte, respectively). The blastocyst formation rates after PA of oocytes matured with 5 and 50ngmL(-1) VEGF were significantly higher (58.99±4.70% and 54.00±1.09%, respectively) than that of oocytes matured with 0 or 500ngmL(-1) VEGF (30.15±4.52%, 34.79±4.01%, respectively). Total cell number of PA blastocyst after oocytes matured with 5 and 50ngmL(-1) VEGF was significantly higher (83.21±4.89, 78.16±6.15, respectively) than that of control and 500ngmL(-1) VEGF (56.91±4.78, 55.93±3.89, respectively). Similarly, the blastocyst formation rate after SCNT of oocytes matured with 5ngmL(-1) VEGF was significantly higher (14.54±1.42%) than that of oocytes matured without VEGF (7.95±1.44%). Total cell number of SCNT blastocyst after oocytes matured with 5ngmL(-1) VEGF was significantly higher (67.83±6.56) than control (48.09±5.36). Fully cumulus cell expansion was significantly higher in the 5ngmL(-1) VEGF treated group (85.37±0.73%) compared with the control (58.89±0.88%). In conclusion, adding 5ngmL(-1) VEGF during IVM improved the developmental potential of PA and SCNT in porcine embryos by increasing the intracellular GSH level.
Reproduction Fertility and Development 01/2011; 23(1):165. · 2.11 Impact Factor
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C H Park,
S G Lee,
H J Lee,
T K Jung,
Y H Jeong,
Y W Jeong, S H Hyun,
Y W Kim,
T Shin,
E-B Jeung,
W S Hwang
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ABSTRACT: It was recently shown that treatment of cloned embryos with histone deacetylase inhibitors improves efficiency for the success rate of developmental potential to term in several species. The objective of the present study was to investigate the influence of the histone deacetylase inhibitor Scriptaid (Sc) on in vitro development in early porcine SCNT embryos and on their gene expression pattern. Based on the findings of previous porcine studies (Zhao et al. 2009), the reconstructed oocytes were treated with 500nM Scriptaid for 14 to 16h after post-fusion activation (6-DMAP/demecolcine). In our preliminary study, blastocyst rate significantly increased in the Sc-treated group, compared with the control group (25.1±2.8% and 13.8±1.9%, respectively, P<0.05). We determined gene expression using quantitative real-time RT-PCR. The results showed that OCT3/4 gene was expressed at a similar level in in vivo and SCNT blastocysts with/without Sc. IGF2 and H19 genes tended to be highly expressed in both SCNT blastocysts with (1.6-fold and 3.1-fold, respectively) and without (2.0-fold and 5.8-fold, respectively) Sc than that of the in vivo blastocysts. We found differences in imprinted gene expression patterns between in vivo and cloned blastocysts. Expression of H19 and IGF2 in SCNT blastocysts after Scriptaid treatment decreased towards the expression levels of in vivo blastocysts. These results indicated that Scriptaid treatment in SCNT embryos may also have beneficial effects on in vitro developmental competence as well as their gene expression pattern.
Reproduction Fertility and Development 01/2011; 23(1):134. · 2.11 Impact Factor
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Y H Jeong,
G H Jang,
I S Hwang,
C H Park,
H J Lee,
Y W Jeong, S H Hyun,
Y W Kim,
T Shin,
E-B Jeung,
W S Hwang
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ABSTRACT: The present study was conducted to establish a porcine transgenic cell line with human CRPs and HT genes, focused on hyperacute rejection (HAR) considering clinical xenotransplantation as alternative sources of human organs. As a first step towards establishing the stable cell line, the cDNA for 3 genes encoding human DAF, CD59, and H-transferase were cloned and sequenced. A tricistronic expression vector was constructed with the aid of 2 IRES elements (pCMV-hDAF_IRES-hHT_IRES-hCD59). The CMV-based expression vector was then introduced into miniature pig ear fibroblast cells by electroporation. Reverse transcription PCR analysis revealed that cell lines stably expressing human transgene-specific transcripts were established. The inhibitory effect of immune response in the established transgenic cell lines was measured by human serum-mediated cytolysis assay, as measured by ELISA. Under the assay conditions (based on human serum from 10 to 50%), the transgenic cell group showed significantly greater survival rate under various serum concentrations than did the nontransgenic cell control group. Moreover, the transgenic cell lines used as nuclear donors for a subsequent NT experiment were confirmed to be expressing their transgene transcripts in vitro developed preimplantation stage embryos. These results indicated that the established cell lines with human transgenes might have an inhibitory effect against lysis by human complement. It is possible that these transgenic cells could serve as nuclear donors to produce transgenic cloned pigs for xenotransplantation.
Reproduction Fertility and Development 01/2011; 23(1):261. · 2.11 Impact Factor
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J Y Lee,
S G Lee,
E J Jung,
S H Jeong,
C J Yang,
Y W Jeong, S H Hyun,
Y W Kim,
T Shin,
E-B Jeung,
W S Hwang
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ABSTRACT: Novel serum-free media (IVD101) has been shown to be effective for the production of in vitro-produced embryos for subsequent implantation into cows (Hoshi 2003 Theriogenology 59, 675-685). The objective of the present study was to determine whether serum-free embryo cultivation during preimplantation stage could be used for the production of bovine transgenic nuclear transfer embryos. Somatic cell nuclear transfer (SCNT) embryos were produced by using donor cells containing a vector to induce the production of human erythropoietin in cow's milk. αS1-casein was selected as the promoter to be used in this study through the specific promoter activity test, and enhanced green fluorescent protein(EGFP) gene was attached to the CMV promoter to allow observation of the donor cell during the experiment. Adult fibroblast cells were transfected with lipofectamine. After G418 selection, the transfected cells were injected to the enucleated oocytes, and injected embryos were accomplished by cell-to-cell fusion. These embryos were then activated with calcium ionomycin and 6-dimethylaminopurine. The reconstructed embryos were cultured in IVD101 and mSOF media at 38.5°C, in a 5% CO(2), 5% O(2), and 90% N(2) atmosphere. Embryos were cultured for 4 days, followed by addition of FBS in case of mSOF media. On day-7, the developmental ability and the number of cells in the reconstructed embryos were determined. Statistical analysis of embryo development data was carried out using unpaired t-test, or ANOVA. There were no significant differences in the cleavage rate (69.6±3.2% v. 64.5±5.0%), blastocyst rate (18.7±1.3% v. 22.0±1.6%), and cell number (113.9±7.5 v. 103.6±7.9) between IVD101 and mSOF+FBS cultured embryos. These results indicated that serum-free media did not reduce the developmental competence of SCNT embryos compared with serum-supplemented media. Further studies are required to investigate whether this serum-free transgenic embryo cultivation could be used for developmental potential in terms of full-term development after embryo transfer.
Reproduction Fertility and Development 01/2011; 23(1):128-129. · 2.11 Impact Factor
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ABSTRACT: Cloning by the process of somatic cell nuclear transfer (SCNT) has been achieved in a variety of mammalian species and has many promising applications. In this process, however, due to reasons beyond current scientific understanding, many results contrary to expectation have also been produced. For instance, abnormal sex development such as demasculinization has been observed in 1 of 6 healthy German shepherd offspring produced with SCNT (1 normal donor (not cloned), 5 cloned but normally developed progenies, 1 cloned sex reversed progeny, and 1 recloned sex reversed progeny from 1 cloned sex reversed progeny). Sex-determining region Y (SRY) is one of the most basic and crucial genes that initiate male sex determination in many mammals. Steroidogenic factor-1 (SF1, NR5A1), which is closely related to SRY, also regulates several genes involved in sex determination. Numerous studies have reported that reduced or deleted SRY gene expression as well as SF1 gene mutations can produced XY sex reversal. To verify the hypothetical association between phenotypic disorder of sex determination and genetic modification by SCNT, we extracted genomic DNA from tissues of normal progeny (not cloned), primary cultured cells of cloned but normally developed progeny, cloned sex reversed progeny, and recloned sex reversed progeny at the age of 1 year and carried out PCR with produced primers based on available SRY and SF1 gene information (SRY gene from AF107021 in GenBank; SF1 gene from ENSCAFG00000023086 in Ensembl). The cloned PCR products were subcloned into T-vector for sequence analysis, which showed no mutation in genetic sequences of SRY and SF1. Taken together, in our case of abnormal sex determination, there was no apparent association between phenotypic sex determination disorder and SRY/SF1 gene mutation. Other sex reversal and related mutation studies have pointed to a wide range of signal networks that include Sox9 and so forth. Further studies should be focused on these other genes in the signal network.
Reproduction Fertility and Development 01/2011; 23(1):239. · 2.11 Impact Factor
