bluuebunny/crossref_metadata_2025 huggingface dataset:
190 EFFECT OF TRANS-ε-VINIFERIN ON IN VITRO PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENTAL COMPETENCE IN PRE-IMPLANTATION EMBRYOS
Authors: Jeon Y. , Kwak S.-S. , Jeong S.-A. , Salehi R. , Seong Y. H. , Hyun S.-H.
Published: December 2011

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 5.0 μM) with 10% porcine follicular fluid, 10 IU mL–1 of eCG and 10 IU mL–1 of hCG. After 22 h in maturation culture, the COC were cultured in hormone-free medium supplemented with various concentrations of trans-ε-viniferin for an additional 22 h 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 5.0 μM) 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 5.0 μM) 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 0.5 μM group (0 μM vs 0.5 μM; 14.6 vs 16.8 pmol oocyte–1) and reduced ROS levels (0 μM vs 0.5 μM and 50 μM; 174.6 vs 25.7 and 23.8 pixel oocyte–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 5.0 μM 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 5.0 μM 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.
This work was supported by a grant from the Korea institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries, Republic of Korea.
192 9-cis-RETINOIC ACID AFFECTS OOCYTE COMPETENCE MARKER GENE EXPRESSION IN THE BOVINE IN VITRO-MATURED OOCYTES AND CUMULUS CELLS
Authors: Deb G. K. , Dey S. R. , Bang J. I. , Kong I. K.
Published: December 2011

Oocyte competence is the ability of an oocyte to undergo pre- and post-implantation development and to deliver a healthy offspring. A close association between the oocyte and the cumulus cells (CC) affects oocyte competence. Expression of several genes in the CC, known as oocyte competence markers, correlates with subsequent embryo development and quality. Addition of 9-cis-retinoic acid (9cisRA) to maturation medium increases oocyte competence through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factors signaling, oxidative-stress protection, or decreasing oocyte TNF-α gene expression. However, the effect of 9cisRA on the expression of oocyte competence markers in the oocytes and CC has not been determined. Therefore, the present study evaluated the effect of 9cisRA on the expression of oocyte competence marker genes in the oocytes and in the CC. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of 0 or 5 nM 9cisRA in the maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 1 μg mL–1 of β-oestradiol, 10 μg mL–1 of follicle stimulating hormone, 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, expression of target transcripts was quantified in CC and zona-free oocytes by SYBER green real-time PCR. The expression was normalized against a minimum of 2 out of 4 reference genes analyzed each time with target genes. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (BioRad) based on M-value during the analysis of gene expression data. A minimum of 5 biological replicates (50–60 oocytes/replicate) were performed for statistical analysis using a Student's t-test. Results indicated that 9cisRA increased (P < 0.05 to P < 0.001) expression of gremlin 1, prostaglandin G/H synthase 2, pentraxin 3, midkine, CD9 and thioredoxin mRNA in oocytes (3.0-, 2.8-, 3.1-, 2.0-, 4.0- and 2.4-fold) and CC (4.0-, 3.2-, 1.9-, 1.7-, 4.0- and 2.4-fold) compared to controls. In contrast, the aldose reductase 1b1 mRNA was down-regulated both in oocytes (1.0- vs 2.0-fold; P < 0.05) and in CC (1.0- vs 1.9-fold; P < 0.04) compared to the respective controls. In conclusion, the present study indicates that 9cisRA influences mRNA expression of oocytes and cumulus cells. This might be another explanation of the improved embryo development and quality in response to 9cisRA during in vitro maturation.
This work was partly supported by a scholarship from the BK21 program, the KRF (KRF-2008-211-F00011), the Next-generation BioGreen 21 Program (No. PJ007990012011), IPET (110020-3 and 109016-3) and the KOSEF (10525010001-05N2501-00110).
193 INSULIN-LIKE GROWTH FACTOR-1 EXERTS A THERMOPROTECTIVE ROLE ON MITOCHONDRIAL FUNCTION OF BOVINE OOCYTES EXPOSED TO HEAT SHOCK
Authors: Ispada J. , Lima R. S. , Risolia P. H. B , Assumpção M. E. O. A. , Visintin J. A. , Paula-Lopes F. F.
Published: December 2011

The series of events associated with oocyte maturation are susceptible to disruption by elevated temperature. These events are regulated by a variety of growth factors, such as insulin-like growth factor-1 (IGF-1). Exposure of bovine oocytes to heat shock compromises oocyte competence and triggers apoptosis. It has been shown that cellular stresses often alter mitochondrial function and activate the mitochondrial apoptotic cascade. Therefore, the objective of this study was to determine the effect of heat shock on bovine oocyte mitochondrial activity and the role of IGF-1 in this context. Slaughterhouse derived cumulus–oocyte complexes (COC) were subjected to control (38.5°C for 22 h) and heat shock (41°C for 14 h, followed by 38.5°C for 8 h) treatments in the presence of 0 or 100 ng mL–1 of IGF-1 during in vitro maturation (IVM). After 22 h, IVM COC were mechanically denuded and subjected to MitoTracker Red CMX-Ros assay (Invitrogen M-7512) to localize and quantify active mitochondria. Denuded oocytes were incubated in TCM-199-HEPES containing 10 μg mL–1 of polyvinyl alcohol and 50 nM MitoTracker at 37°C for 15 min. Oocytes were evaluated under fluorescence microscope and digital images were obtained and stored as TIFF files. Mitochondrial activity from each oocyte was quantified using the software Image J 1.43. This experiment was replicated 6 times using 97 to 204 COC/treatment. Data were analyzed by least-squares analysis of variance using the general linear model procedure of SAS. In the absence of IGF-1, heat shock reduced (P < 0.001) mitochondrial activity from 64.31 ± 1.91 to 56.74 ± 1.26 arbitrary units for control and heat shock groups, respectively. Addition of IGF-1 to maturation medium did not affect mitochondrial activity in the control group (66.25 ± 1.56). However, IGF-1 improved (temperature × IGF-1; P < 0.001) mitochondrial activity of bovine oocytes subjected to heat shock (70.32 ± 1.32). In conclusion, heat shock reduced bovine oocyte mitochondrial activity, suggesting activation of mitochondrial apoptotic cascade. Moreover, IGF-1 exerted a thermoprotective role, reducing the mitochondrial damage caused by elevated temperature.
194 IN VITRO MATURATION AND RNA CONTENT AND DISTRIBUTION OF PORCINE OOCYTES DERIVED FROM SMALL AND MEDIUM FOLLICLES AND CLASSIFIED BY BRILLIANT CRESYL BLUE ASSAY
Authors: Ngoc Thanh L. T. , Funahashi H.
Published: December 2011

Oocytes collected from the surface of slaughterhouse ovaries clearly have heterogeneous quality. The objective was to characterize a change or difference in RNA distribution and the content of porcine oocytes that were collected from small (SF; 1–2 mm in diameter) and medium follicles (MF; 3–6 mm in diameter) and assessed for glucose-6-phosphate dehydrogenase (G6PD) activity by brilliant cresyl blue (BCB) assay. Following BCB staining, porcine cumulus–oocyte complexes (COC) with dark blue (DB; G6PD-inactive, suggesting good quality) and light blue (LB; G6PD-active, suggesting immaturity) ooplasm were then separately cultured in vitro to evaluate nuclear maturation and analyze the characteristics of the RNA aspect. RNA distributions in cumulus cell mass and ooplasm were labeled with fluorescence, SYTO RNA select green and then examined at different periods of culture for in vitro maturation (IVM; with gonadotropins and dbcAMP for 20 h and then without those for 24 h). Total RNA content of oocytes and cumulus cell mass were measured by using a manufacture's kit (Invitrogen Quant-iT RNA assay kit). Statistical analyses of results from 3 to 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P < 0.05). When oocytes were classified by BCB assay, the percentage of oocytes with DB cytoplasm was much higher (P < 0.05) in oocytes from MF (72.5% of 208) compared with those of SF (53.6% of 352). Regardless of the origin of oocytes (SF vs MF), the incidence of mature oocytes following culture was higher in DB than LB (64.0% of 147 and 72.1% of 133 vs 50.9% of 108 and 52.8% of 54, respectively). Twenty hours after the start of IVM, the DB oocytes had a higher proportion of RNA-free zone inside the germinal vesicle compared to the LB oocytes (66.1% of 193 and 26.8% of 159 in MF; 47.9% of 185 and 17.9% of 184 in SF; P < 0.05). When the total content of RNA was examined during IVM, both oocytes from SF and MF contained higher levels of total RNA at the beginning of IVM as compared with 20 and 44 h after the start of IVM, whereas the content (40 denuded oocytes/sample) was higher in oocytes with DB cytoplasm from MF than those from SF (P < 0.05). Regardless of the origin of oocytes from SF and MF, the total content of RNA in oocytes with LB cytoplasm was significantly lower than in oocytes with DB cytoplasm (P < 0.05) before the start of IVM. The total RNA content of cumulus cells (40 COC/sample) before IVM culture was also higher in the cell mass surrounding the DB oocytes than the LB ones and in the cell mass surrounding the oocytes from MF rather than SF (P < 0.05). These results demonstrate a possibility that differences in RNA distribution and content in oocytes, as well as the content in cumulus cells, reflect in the ability of porcine oocytes to mature in vitro.
20 ALTERED GENE EXPRESSION IN BOVINE SOMATIC CELL NUCLEAR-TRANSFERRED EMBRYOS AFTER TRICHOSTATIN A TREATMENT
Authors: Camargo L. S. A. , Pereira M. M. , Wohlres-Viana S. , Quintão C. R. C. , Iguma L. T. , Carvalho B. C. , Siqueira L. G. B. , Viana J. H. M.
Published: December 2011

Trichostatin A is a histone deacetylase inhibitor that improves histone acetylation and chromatin remodeling of somatic cell nuclear-transferred embryos (Iager et al. 2008 Cloning Stem Cells 10, 371–379; Maalouf et al. 2009 BMC Dev. Biol. 9, 11). We have previously observed that it also improves quality of bovine cloned embryos, which may increase pregnancy rates. This study aimed to evaluate the effect of trichostatin A treatment of zygotes on relative abundance of 9 transcripts in bovine nuclear-transferred blastocysts. In vitro matured oocytes were enucleated, fused to somatic cells and activated with ionomycin (Camargo et al. 2011 Reprod. Fertil. Dev. 23, 122). After activation, putative zygotes were randomly separated into 2 groups: NT-TRICHO, zygotes were cultured for 4 h in 6-DMAP followed by 7 h in CR2 aa medium plus with 2.5% fetal calf serum (FCS; Nutricell, Campinas, Brazil), both supplemented with 50 nM trichostatin A (Sigma); NT-CONT, zygotes were cultured in the same described conditions without thichostatin A supplementation. In vitro-fertilized embryos (IVF group) were used as a calibrator for relative transcript quantification. Embryos from the 3 groups were cultured in CR2 aa supplemented with 2.5% FCS under 5% CO2, 5% O2 and 90% N2 at 38.5°C. At 168 h postactivation, the embryos were rapidly frozen in liquid nitrogen. Pools of 10 blastocysts for each group were subject to RNA extraction and reverse transcription, in which cDNA was amplified by real-time PCR using the β-actin and GAPDH genes as endogenous references. The transcripts analysed encode high mobility group N1 (HMGN1), peroxiredoxin 1 (PRDX1), octamer-binding protein 4 (OCT4), insulin-like growth factor 1 and 2 receptors (IGF1r and IGF2r), glucose transporter 1 and 5 (GLUT1 and GLUT5), histone acetyltransferase (HAT) and heat shock protein 70.1 (HSP70) genes. Results were analysed by a pair-wise fixed reallocation randomization test using the REST software v.2. Data from NT-TRICHO and NT-CONT were compared with the IVF group and between themselves. The relative abundance of HSP70, PRDX1, IGF2r and HMGN1 transcripts was higher (P < 0.05) in NT-TRICHO compared with the IVF group and no difference was detected for the other transcripts. In the NT-CONT group, the relative abundance of IGF2r and HAT was higher (P < 0.05), whereas IGF1r and OCT4 were lower (P < 0.05) compared with IVF embryos. When data from NT-TRICHO and NT-CONT were compared, a higher amount (P < 0.05) of stress-associated transcripts (HSP70 and PRDX1) were found in NT-TRICO blastocysts. These results suggest that although trichostatin A may improve chromatin remodeling, alterations on gene expression still persist in bovine somatic cell nuclear-transferred blastocysts in comparison with IVF embryos.
Financial support: Embrapa Project 01.07.01.002, CNPq 403019/2008–7 and Fapemig.
206 GENERATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS FROM DENTAL PULP-DERIVED MESENCHYMAL STEM CELLS
Authors: Lee J. H. , Lee Y. M. , Maeng G. H. , Jeon R. H. , Kim T. H. , Lee W. J. , Ock S. A. , Jeon B. G. , Mohana Kumar B. , Lee S. L. , Rho G. J.
Published: December 2011

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state and a great source for regenerative medicine. Several types of human somatic and adult stem cells have been reprogrammed into iPS cells, including mesenchymal stem cells (MSC). Recently, human dental pulp has been considered as a valuable alternative source of MSC (hDP-MSC) with excellent proliferation capacity and multilineage differentiation potential. In this study, our objective was to establish iPS cells from hDP-MSC and evaluate the expression of transcriptional factors and in vitro differentiation potential into mesenchymal lineages. The hMSC were isolated from the dental pulp of male donor (∼18 years old) were cultured in advanced-DMEM supplemented with 10% fetal bovine serum at 37°C, 5% CO2 in a humidified atmosphere. The hDP-MSC at passage 3 were analysed for the expression of MSC-specific surface markers (CD44 and CD90) using flow cytometry and transcriptional factors (Oct4, Nanog and Sox2) by immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR). Differentiation into adipocytes and osteocytes of hDP-MSC was carried out under specific conditions for 2 and 4 weeks, respectively and assessed by cytochemical staining (Oil red O, von Kossa and Alizarin Red S, respectively). iPS cells were generated from hDP-MSC at passage 3 by using pMXs retroviral vector (Addgene, Cambridge, MA, USA) containing cDNA of c-Myc, Klf4, Nanog and Sox2. The iPS cells were evaluated for alkaline phosphatase (AP) activity, expression of human embryonic stem cells (hESC) markers (Rex1, Nanog, Oct4, SSEA-1 and TRA-160) by immunostaining. Isolated hDP-MSC expressed surface markers, such as CD44 and CD90 (86% and 93%, respectively) by flow cytometry and positively stained for transcriptional factors (Oct4, Nanog and Sox2) by immunofluorescence. Further, the cells were capable of differentiating in vitro into adipocytes and osteocytes as demonstrated by Oil red O and von Kossa and Alizarin red S staining, respectively. The iPS cells generated from hDP-MSC were positive for AP staining and clearly expressed the markers specific to hESC, including Rex1, Nanog, Oct4, SSEA-1 and TRA-160. In conclusion, hMSC derived from dental pulp could be successfully reprogrammed into iPS cells by retroviral vector systems and the generated iPS cells shared the similar characteristics of hESC. Therefore, hDP-MSC might be an ideal alternative cell source to derive autologous iPS cells for therapeutic applications.
This work was supported by Grant No. 2007031034040 from Bio-organ and Grant No. 200908FHT010204005 from Biogreen21.
207 ENGINEERED HUMAN AMNIOTIC FLUID-DERIVED STEM CELLS BY EXPRESSING CYTOSINE DEAMINASE (CD) AND THYMIDINE KINASE (HSV-TK) MEDIATE TARGETING KILLING EFFECT IN BREAST CANCER CELLS
Authors: Kang N.-H. , Choi K.-C.
Published: December 2011

Genetically engineered stem cells (GESTECs) expressing suicide genes which can convert non-toxic prodrugs to toxic metabolites have been shown to reduce tumour growth. In this sudy, we employed human amniotic fluid-derived stem cells (hAFSCs) as a source of cells for suicide gene transfer. hAFSCs were engineered to express multiple suicide genes, a bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) genes. Thus, AF2.CD-TK cells were generated to express CD and HSV-TK genes simultaneously, which can convert 5-fluorocytosine (5-FC) and monophosphorylate ganciclovir (GCV-MP) into 5-fluorouracil (5-FU) and active form of triphosphate ganciclovir (GCV-TP), respectively. Combination of CD and HSV-TK suicide genes in AF2.CD-TK cells resulted in enhanced antitumour activity in vitro and in vivo xenograft models. AF2.CD-TK cells significantly inhibited the growth of MDA-MB-231 human breast cancer cells in the presence of the prodrugs, 5-FC and GCV, by MTT assay. MDA-MB-231 cells were implanted into the right mammary fat pad of female BALB/c nude mice and CM-DiI-labelled AF2.CD-TK cells were injected into the circumtumoral site with 5-FC (500 mg kg–1 day–1) and GCV (10 mg kg–1 day–1). Treatment of the mice with AF2.CD-TK cells significantly reduced tumour volumes in MDA-MB-231 xenografted mice. We further evaluated tumour progression by histopathological and fluorescent staining. Taken together, these results indicate that AF2.CD-TK cells can serve as a vehicle for a novel therapeutic approach enzyme/prodrug system to selectively target breast malignancies.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0015385).
21 EFFECT OF SECOND TIME XENOPUS EGG EXTRACT TREATMENT ON COLONY FORMATION AND CLONED BLASTOCYST FORMATION IN PIG
Authors: Liu Y. , Østrup O. , Li R. , Vajta G. , Kragh P. M. , Purup S. , Callesen H.
Published: December 2011

Extract from Xenopus eggs can induce reprogramming in somatic cells. In our previous study, cell colony formation was induced during culture of porcine fetal fibroblasts after a single treatment with Xenopus egg extract and culture for several passages and using these long-term cultured cells for cloning increased the resulting blastocyst rate (Liu et al. 2011 Reprod. Fertil. Dev. 23, 130). However, both colony number and cloned blastocyst rate decreased after Passage 15 and no colonies formed after Passage 18. Therefore, in this study we investigated the effect of a second extract treatment on colony formation and cloned blastocyst formation. Extract-treated (ExT) porcine fetal fibroblasts at Passage 13 (P13) grown on poly-L-lysine-coated coverslips were permeabilized by digitonin (7 μg mL–1, 2 min, 4°C) and incubated in extract at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445). The treated cells were split onto 2 coverslips on Day 7 after the second extract treatment (2ExT), defined as Passage 1 (2ExT P1, comparable with ExT P14). New subcultures were made every 7 to 8 days when 70 to 80% clusters became colonies (i.e. 2ExT P8). Colony cells from both ExT (P14 and P16) and 2ExT (P1, P3 and P6) were used for handmade cloning and nontreated cells were used as control (Day 0). Blastocyst rates were analysed by chi-square test and colony numbers were analysed by 1-way ANOVA (SAS version 9.2). Colony numbers and cloned blastocyst rates on Day 6 are summarised in Table 1. Colonies continued to form in treated cells from 2ExT P1 to P8. The colony number maintained at a high level (60 to 80) from 2ExT P4 to P8 and it was significantly higher than that of ExT cells at the comparable passage numbers. No colonies formed in control cells. When using 2ExT colony cells at P3 and P6 for cloning, the blastocyst rates increased compared with controls and they were also higher than in the ExT group. Cloned blastocyst rates were not different between 2ExT P1 and ExT P14 groups. In conclusion, a second extract treatment can induce colony formation and increase cloned blastocyst rates, indicating that this repeated extract treatment again could activate the extract-treated cells to an activity level similar to that achieved after the first treatment.

Table 1.Summary of colony number and cloned blastocyst rate with ExT and 2ExT colony cells
216 EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS ANALOGOUS TO INNER CELL MASS-DERIVED LIF-DEPENDENT MOUSE EMBRYONIC STEM CELLS ESTABLISHED FROM THE DOMESTIC PIG, SUS SCROFA
Authors: Telugu B. P. , Ezashi T. , Alexenko A. , Lee S. , Prather R. S. , Roberts R. M.
Published: December 2011

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer.
Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.
217 PROTEIN-INDUCED TRANSDIFFERENTIATION INTO MALE GERM CELL-LIKE LINEAGE FROM CHICKEN FETAL BONE MARROW STEM CELLS
Authors: Yoo J. M. , Park J. J. , Gobianand K. , Ji J. Y. , Kim J. S. , Song C. S. , Song H. , Byun S. J. , Park J. K. , Yoo J. G.
Published: December 2011

Bone marrow (BM)-derived stem cells are capable of transdifferentiation into multilineage cells like muscle, bone, cartilage, fat and nerve cells. In this study, we investigated the capability of mesenchymal stem cells (MSC) derived from BM into germ cell differentiation in the chicken. Chicken MSCs were isolated from BM of day 20 fertilized fetal chicken with Ficoll-Paque Plus. Isolated cells were cultured in advance-DMEM (ADMEM) supplemented with 10% fetal bovine serum and antibiotics. Once confluent, cells were subcultured until five passages. The cultured cells showed fibroblast-like morphology. The cells had positive expressions of Oct4, Sox2 and Nanog. Two induction methods were conducted to examine the ability of transdifferentation into male germ cells. In group 1, MSC were cultured in ADMEM containing retinoic acid and chicken testicular extracts proteins for 10 to 15 days. In group 2, MSC were permeabilized by streptolysin O and treated with chicken testicular protein extracts. In both treatment groups, MSC were cultured in ADMEM containing retinoic acid for 10 to 15 days. We found that chicken MSC had a positive expression of pluripotent proteins such as Oct4, Sox2, Nanog and a small population of chicken MSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers and male germ-cell-specific markers (Dazl, C-kit, Stra8 and DDX4) as analysed by reverse transcription-PCR and immunohistochemistry. These results demonstrated that chicken MSC may differentiate into male germ cells and the same might be used as a potential source of cells for production of transgenic chickens.
This study was carried out with the support of Agenda Program (Project No. PJ0064692011), RDA and Republic of Korea.
235 PRODUCTION OF PORCINE KNOCK-IN FIBROBLASTS EXPRESSING HUMAN DECAY-ACCELERATING FACTOR on α-1,3-GALACTOSYLTRANSFERASE (GGTA1) GENE LOCUS
Authors: Kim J. W. , Lee S. M. , Ju J. W. , Kang M. J.
Published: December 2011

Galactose-α1,3-galactose (α1,3-gal) epitope is synthesised at a high concentration on the surface of pig cells by α1,3-galactosyltransferase gene (GGTA1). The α1,3-gal is responsible for hyperacute rejection in pig-to-human xenotransplantation. The generation of transgenic pigs as organ donors for humans is necessary to eliminate the GGTA1 gene that synthesise Galα(1,3)Gal. Knock-in system refers to the integration a foreign gene at a specific target, disrupting the target gene by inserting the transgene. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as complement inhibitor to protect self cells from complement attack. The generation of GGTA1 knockout pigs expressing DAF is necessary to use as organ donors for humans. In this study, we established cell lines of GGTA1 knockout expressing DAF from pig ear fibroblasts to apply somatic cell nuclear transfer. A GGTA1-DAF knock-in vector for positive-negative selection (PNS) consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the diphtheria toxin A fragment (DT-A) gene as a negative selectable marker gene. The DAF gene was subcloned in the GGTA1 gene exon 4 and the endogenous start codon was replaced by that of DAF. The ear fibroblast cell lines were isolated from Chicago miniature pig and used for transfection. Linearized targeting vector DNA was transfected into these cells by electroporation. After selection of G-418 during 11 days, we analysed 242 colonies using PCR and long PCR. We isolated 2 positive colonies for GGTA1 gene disruption. To confirm expression of the hDAF and GGTA1 genes, we isolated total RNA from normal pig cells and the knock-in somatic cell line. A cDNA synthesised using reverse transcriptase progressed to RT-PCR by a pair of DAF-specific primers and α1,3-GT specific primer, respectively. Expression of DAF was detected in DAF knock-in somatic cells, butnot in normal pig cell. Expression of GGTA1 gene was decreased in the knock-in somatic cell line compared with normal pig cell. These cell lines may be used production of α1,3-GT knockout and DAF expression pig for xenotransplantation.
237 EXPRESSION PATTERNS OF DEUBIQUITYLATING FACTORS (Usp9x AND Af-6) IN GERM CELLS OF WILD TYPE AND TRANSGENIC PIGS
Authors: Oh M.-Y. , Shim M.-R. , Cho Y.-M. , Kim K.-Y. , Lee H.-C. , Yang B.-C. , Chung H.-K. , Park C.-K. , Park J.-K. , Chung H.-J.
Published: December 2011

The ubiquitin (ub)-mediated degradation of certain regulatory proteins plays critical roles in various functions, including cell cycle, signal transduction, transcription regulation and endocytosis. Usp9x is stage dependently expressed in the germ cells during mouse gametogenesis. Af-6, a cell junction protein, has been identified as a substrate of Usp9x, indicating a possible association between Usp9x and Af-6 in spermatogenesis and oogenesis. In this study, we examined the expression patterns of Usp9x and Af-6 and their intracellular localization in transgenic pig testes and ovary. In both Sertoli and granulosa cells, Af-6 was continuously expressed throughout postnatal and adult stages. Both Af-6 and Usp9x were enriched at the sites of Sertoli-spermatid junctions, especially at stage IV. In granulosa cells, Usp9x was strongly expressed in primary follicles, but its expression rapidly decreased after the late-secondary follicle stage. On the other hand, the expression of both Usp9x and Af-6 was weak in transgenic pigs, with low expression in the Sertoli cells and relatively stronger expression in Graafian follicles. This study suggests that Usp9x and Af-6 were deubiquitylated in Sertoli and follicle cells in wild type and transgenic pigs.
This study was carried out with the support of Agenda Program (Project No. PJ006702), RDA, Republic of Korea.
239 A NOVEL PIG MODEL OF CYSTIC FIBROSIS GENERATED BY SEQUENTIAL TARGETING OF CFTR BY BACTERIAL ARTIFICIAL CHROMSOME VECTORS
Authors: Klymiuk N. , Mundhenk L. , Wallner K. , Wuensch A. , Richter A. , Plog S. , Stehr M. , Holzinger A. , Kröner C. , Kessler B. , Kurome M. , Zakhartchenko V. , Nagashima H. , Gruber A. D. , Wolf E.
Published: December 2011

Cystic fibrosis (CF), the most common inherited disease in the Caucasian population, is caused by mutations of the CFTR gene, coding for an anion channel responsible for chloride movement, determining transepithelial salt transport, fluid flow and ion concentrations. Cystic fibrosis is determined by chronic lung disease, marked by airway surface dehydration, reduced mucus clearance and increased mucus obstruction, impaired bacterial killing and inflammation and continuous destruction of the lung tissue. The lack of suitable CF mouse models has so far impeded the understanding of disease mechanisms and the development of novel therapies. Recently, a pig model reflecting the main hallmarks of the human disease in the respiratory, gastrointestinal and genital tracts has been reported (Rogers et al. 2008 Science 321, 1837–1841). In the present study, we generated a new CF pig model by disrupting the CFTR gene using an approach that differs in 3 major aspects from the previously published model: (1) gene targeting was achieved using a modified bacterial artificial chromosome (BAC) vector; (2) transcription of the CFTR gene was completely prevented; and (3) homozygous CFTR mutant pigs were derived by nuclear transfer from cells in which both alleles were inactivated by sequential BAC targeting. We characterised 2 cloned litters with a total of 13 piglets (including 4 stillborns). All piglets showed a characteristic CF multi-organ disease with severe meconium ileus, microcolon, microgallbladder, hypoplasia of the exocrine pancreas, congenital aplasia of the vas deferens and malformations of the trachea. In contrast to the recently published CF pig, where the majority of the intestinal obstructions occurred proximal to the ileocecal junction, we found obstructions exclusively distal to this site. We thus present a novel pig model for CF that reflects the main hallmarks of the human disease. It confirms the suitability of the CFTR mutant pig for cystic fibrosis research and increases the availability of CF models for molecular and translational CF research.
The help by Prof. K. Heinritzi and his team from the Pig Clinic, LMU Munich, is gratefully acknowledged. This study was supported by Mukoviszidose e.V.
ANIMAL MODELS FOR THE STUDY OF HUMAN OVARIAN FUNCTION
Authors: Adams G. P. , Baerwald A.R.
Published: December 2011

The study of ovarian function has been of great interest to scientists and clinicians in the human and veterinary health fields for more than 20 centuries. Initial studies of the ovaries were based on anatomical descriptions post-mortem, followed by histological and endocrinological approaches. The introduction of high-resolution ultrasonography provided a long-awaited tool to image the reproductive tissues in situ in both animals and humans. Critical studies of the characteristics and control of ovarian follicular and luteal dynamics in non-human primates, rodents, and domestic farm animals have involved frequent (i.e. daily or multiple times a day) blood sampling and ultrasonography. Studies of this nature in women are difficult, and often unethical to conduct. Differences in antral folliculogenesis between humans and animals appear to be more in detail rather than in essence, and may reflect differences in intrinsic physiology or merely differences in our ability to detect changes in a given species. In women, the presence of endometrial shedding and symmetric luteal and follicular phases are different from that observed during the estrous cycles of domestic farm animals but despite these differences, general similarities in antral follicular dynamics exist. A continuous pattern of antral follicle development was originally proposed in domestic livestock species; however, the use of frequent serial ultrasonography and simultaneous endocrine profiling in these animal species has resulted in a broad understanding of follicular wave dynamics. Follicular waves have now been described in every species in which this approach has been used, including humans. The relatively large diameters of antral follicles in cows and mares, compared to monkeys, sheep, and rodents provide greater feasibility for characterizing antral follicular dynamics ultrasonographically. Profound similarities in the dynamics of follicle development exist between the menstrual cycle in humans and the estrous cycle in cattle and horses. Despite a proportionately longer luteal phase in cows and mares, the growing phase of the dominant ovulatory follicle (defined as the time period from wave emergence to ovulation) is comparable in the human, cow, and mare. Thus, at present, cows and mares may be the most appropriate models for the study of antral follicular dynamics in women. While the use of animal models has increased our understanding of ovarian function and provides the hypothetical basis for studies in women, differences in vocabulary, culture, and research methodologies between scientists in predominantly human versus animal settings has hampered knowledge translation. These differences represent a systemic impediment to a broad understanding of ovarian function and limits progress and innovation in the development of safer and more efficacious treatments for infertility and contraception.
GENERATION OF RABBIT PLURIPOTENT STEM CELL LINES
Authors: Dinnyes A. , Pirity M. K. , Gocza E. , Osteil P. , Daniel N. , Tancos Zs. , Polgar Zs. , Maraghechi P. , Ujhelly O. , Nemes C. , Stout T. , Tapponnier Y. , Bosze Zs. , Jouneau A. , Afanassieff M. , Savatier P.
Published: December 2011

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in the rabbit. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit full pluripotency in vivo. Moreover, similarly to several domestic species, markers used to characterize the putative ESCs are not fully adequate because studies in domestic species have revealed that they are not specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing rabbit embryo. The status of isolation and characterization of the putative pluripotency genes in rabbit will be discussed. Using rabbit specific pluripotency genes we might be able to reprogram somatic cells and generate induced pluripotent stem cells more efficiently thus overcome some of the challenges towards harnessing the potential of this technology.
This study was financed by EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; RabPstem, PERG07-GA-2010-268422; PluriSys, HEALTH-2007-B-223485; AniStem, PIAP-GA-2011-286264), NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; Plurabbit, OMFB-00130-00131/2010 ANR-NKTH/09-GENM-010-01.
THE HORSE AS A LARGE ANIMAL MODEL FOR EXTRA-FETAL DERIVED CELL THERAPY OF CHRONIC RESPIRATORY DISEASES
Authors: Consiglio Anna Lange , Zucca Enrica , Cremonesi Fausto , Laverty Sheila , Lavoie Jean Pierre , Ferrucci Francesco
Published: December 2011

The airway epithelium is subjected to a lifetime exposure to inhaled particles and pathogens that may lead to the development of a variety of infectious and inflammatory respiratory diseases such as chronic bronchitis, asthma and chronic obstructive pulmonary disease. These disorders are typically associated with changes in the architecture of the airway walls,that range from epithelial structure remodeling to complete denudation of the basement membrane. The repair of injuries and the regeneration of the epithelial structure involve stem and progenitor cells. Although both secretory and basal cells are able to proliferate, only basal cells are recently suggested to represent the stem cell (SC) niche of the airway epithelium in human tracheas and bronchi, but the adult secretory cells lose their regeneration potential compared to the fetal secretory cells. For this reason, researchers are considering other sources for exogenous pluripotent SCs for airway tissue engineering. At present, autologous bone marrow and adipose derived MSCs seem to present the most popular SC type used in laryngotracheal tissue engineering. Extra-fetal derived SCs could represent new alternative cell sources for lung regeneration. Investigations of stem cell therapy for murine lung injuries revealed an excellent regeneration potential of extra-fetal derived cells that integrated into the lung and differentiated into pulmonary lineages after injury. Recurrent airway obstruction disease (RAO) in the horse is one of the only naturally occurring diseases in animals that is comparable to bronchial asthma in humans. The anamnestic and reversible nature of equine RAO is similar to some forms of human asthma suggesting a common immunological basis. Based on similarities between human asthma and equine RAO, we propose to use spontaneously RAO affected horses as an animal model in biomedical research. We describe the isolation, in vitro proliferation capacity and labeling of equine extra-fetal derived cells, and preliminary in vivo results indicating that after local injection, labeled cells could be retrieved by bronchoalveolar lavage from selected pulmonary areas.
REPROGRAMMING OF PORCINE EPIBLAST-DERIVED NEURAL PROGENITOR CELLS TO PLURIPOTENCY
Authors: Rasmussen M. A. , Hall V. J. , Petkov S. G. , Ujhelly O. , Pirity M. , Dinnyes A. , Niemann H. , Hyttel P.
Published: December 2011

Human induced pluripotent stem cells (iPSC) and neural progenitor cells (NPC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC and NPC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 ml active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC, and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC, and pKLF4 (pOSMK); all including 0.25 ml transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL–1 human bFGF and 2 μg mL–1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK, and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and sub-cultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC, and KLF4, as evaluated by immunocytochemistry, and expression of NANOG, SSEA-1, and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR, and 150 ng mL–1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The latter resulted in establishment of new NPC lines. The system can be used to study mechanisms involved in the early transition from pluripotency to multipotency in the pig and the reversal of the process caused by reprogramming.
The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 Stem Cell Project “PartnErS” (218205; 204, 523) and EU FP7 Stem Cell Project “PluriSys” (223485).
41 THE EFFECT OF AMBIENT TEMPERATURE ON SPERM MOTILITY DURING LIQUID STORAGE OF VENDA COCK SEMEN AND INDIVIDUAL DIFFERENCES IN SPERM CRYOTOLERANCE
Authors: Mphaphathi M. L. , Luseba D. , Masenya M. B. , Sutherland B. , Nedambale T. L.
Published: December 2011

Improving techniques for liquid storage of cock semen can increase the efficiency of AI programs in the poultry industry. The aims of the present study were (1) to compare storage of cock sperm for 24 h at 5 and 25°C and (2) to test the cryotolerance of sperm cell motility in individual Venda cocks. Semen was collected with the abdominal massage method, from 6 indigenous Venda cocks. Cocks were 26 weeks of age and were kept under the same conditions. After macroscopic analysis, semen was pooled and diluted (1:2) with Kobidil+ extender and divided into 3 equal parts. Part 1 was evaluated immediately (0 h), part 2 was stored at 5°C and part 3 was stored at 25°C and evaluated for sperm motility and velocity parameters at 4, 8, 12 and 24 h of storage. For cryopreservation, semen was diluted (1:2) with modified Kobidil+ extender supplemented with 8% of dimethyl sulfoxide. Individual ejaculates were equilibrated at 5°C for 4 h and then loaded into the programmable freezer. Then, semen straws were thawed at 5°C. Sperm motility and velocity parameters were evaluated using the Sperm Class Analyzer® system. Six replicates were done per trial. Data were analysed using the statistical programme GenStat®. Treatment means were separated using Fisher's protected t-test least significant difference (P < 0.05). Total sperm cell motility rate was 87.5% and decreased significantly during in vitro storage and was <31% after 24 h at 25°C. Semen samples stored at 5°C showed a total sperm cell motility rate of above 50% after 24 h. There was a slight linear decrease in the percentage of sperm with progressive motility and rapid velocity as the storage period increased, irrespective of the storage temperature. The rapid and medium motility percentages were higher in fresh semen and significantly decreased (P < 0.05) during the incubation period. There was variation in the total sperm cell motility of fresh and frozen semen among cocks. There was no significant difference in variation in non-progressive and medium percentage (P > 0.05) motility in diluted fresh or frozen sperm cells or in the percentage of sperm with rapid motility in thawed semen. There was variation in <25% of the cocks in total sperm motility rate. In summary, cryopreservation reduced sperm cell motility and velocity rates in all the cock semen donors. We found that cryotolerance of cock sperm does vary among males. Furthermore, the lower temperature 5°C was suitable for semen storage of Venda cocks. This temperature (5°C) could potentially improve methods of semen equilibration before cryopreservation.
The study was supported by an Agricultural Research Council Parliamentary Grant, Department of Agriculture Forestry and Fisheries and National Research Foundation-GUN No RT21 and 24000 (NRF). The Germplasm Conservation & Reproduction Biotechnologies (GCRB) group is thanked for their support.