16 t h International Congress on Animal <strong>Reproduction</strong> 202 Poster Abstracts P528 Development of reconstructed embryos from human somatic cells and ov<strong>in</strong>e enucleated oocytes and isolation of putative human embryonic stem cell clones Zhou, MH*; Zhao, J Faculty of Bioeng<strong>in</strong>eer<strong>in</strong>g, Inner Mongolia Agricultural University, Ch<strong>in</strong>a Introduction S<strong>in</strong>ce the difficulty of obta<strong>in</strong><strong>in</strong>g human embryos limited the production of human embryo stem cells this research was to clone heterogeneous embryos by us<strong>in</strong>g human fetal sk<strong>in</strong> fibroblast cells as nuclear donor cells and the enucleated ov<strong>in</strong>e oocytes as recipient cytoplasts for exam<strong>in</strong><strong>in</strong>g the <strong>de</strong>velopmental potential of the reconstructed embryos to attempt to isolate human embryonic stem cells. Methods Ov<strong>in</strong>e oocytes were collected from slaughterhouse-<strong>de</strong>rived ovaries, <strong>in</strong> vitro matured and enucleated then used as recipient cytoplasm. Human fibroblast cells was obta<strong>in</strong>ed from an aborted 4- month aged fetus sk<strong>in</strong> and employed as donor somatic cells. The cell was <strong>in</strong>jected to sub-zona pellucida of oocyte and fused electrically <strong>in</strong>to ooplasm then activated by Inomyc<strong>in</strong> with 2 M/ml 6- dimethylam<strong>in</strong>opur<strong>in</strong>e (6-DMAP). Only the activated reconstructed embryos were co-cultured with ov<strong>in</strong>e cumulus cells <strong>in</strong> synthetic oviduct fluid supplemented with am<strong>in</strong>o acid (SOFaa) and 10% (v/v) fetal calf serum (FCS). These heterogeneously cloned embryos were morphologically monitored for their <strong>de</strong>velopmental competence at 48h to 168 h of culture. The cell mass of morula or blastocysts were cultured on mouse fibroblast fee<strong>de</strong>r layer and used for the isolation of embryonic stem cell clones. The clones of putative human embryonic stem cell were i<strong>de</strong>ntified by alkal<strong>in</strong>e phosphatase (AKP). Results The human fetal sk<strong>in</strong> fibroblast cells ma<strong>in</strong>ta<strong>in</strong>ed morphologically normal characteristics and normal numbers of chromosomes (2n = 64) <strong>in</strong> culture. The reconstructed human embryos completed the first cleavage of between 24 to 48 h and morula/blastocyst <strong>de</strong>velopment <strong>in</strong> 72 h to 168 h after activation. 27% of the reconstructed embryos un<strong>de</strong>rwent the cleavage while only 15% of the cleaved embryos procee<strong>de</strong>d to <strong>de</strong>velop to morula/blastocyst. The clones of putative embryonic stem cell grew on fibroblast cell layer, with nest- or islet-like morphology after 3 to 7 days culture. AKP sta<strong>in</strong><strong>in</strong>g of the clones showed positive reaction. Conclusions Human fetal fibroblast cell nuclei can be reprogrammed <strong>in</strong> ov<strong>in</strong>e enucleated oocytes. The heterogeneously nuclear-transferred human embryos can un<strong>de</strong>rgo the meiosis division and subsequent <strong>de</strong>velopment to morula/blastocyst stage thus serv<strong>in</strong>g as an alternative for obta<strong>in</strong><strong>in</strong>g human embryonic stem cells. The improvement of the reconstructed embryo <strong>de</strong>velopmental ability, however, is a future challenge. Poster 19 - Biomedical Mo<strong>de</strong>ls <strong>in</strong> Reproductive and Regenerative Medic<strong>in</strong>e P529 Embryonic Stem Cells <strong>de</strong>rived from Separated Blastomeres of Cynomolgus Monkey as a Mo<strong>de</strong>l System of Regenerative Medic<strong>in</strong>e Hosoi, Y 1 *, Teramura, T 2 , Takenoshita, M 3 , Takehara, T 1 , Matsumoto, K 1 , Saeki, K 1 , Fukuda, K 2 , Iritani, A 1 1Dept of Genetic Eng<strong>in</strong>eer<strong>in</strong>g, K<strong>in</strong>ki University, Japan; 2 Dept of Orthopedic Surgery, K<strong>in</strong>ki University, Japan; 3 Keari Co. Ltd., Japan Objective Recently, ESCs (Embryonic Stem Cells) were established from s<strong>in</strong>gle blastomeres of preimplantation embryos <strong>in</strong> mice and humans. These methods have been suggested to reduce ethical concerns s<strong>in</strong>ce we can obta<strong>in</strong> pluripotent ESCs without <strong>in</strong>terfer<strong>in</strong>g embryonic <strong>de</strong>velopment. Furthermore, these results also give rise to important <strong>in</strong>formation about polarity of mammalian embryos. Although totipotencies of separated blastomeres have been experimentally <strong>de</strong>term<strong>in</strong>ed <strong>in</strong> ro<strong>de</strong>nts and some domestic animals, only one live-birth was reported <strong>in</strong> primate. Here, we succee<strong>de</strong>d <strong>in</strong> establishment of ESCs from s<strong>in</strong>gle blastomeres of two-cell and fourcell embryos <strong>in</strong> cynomolgus monkey. Methods In this study, we cultured the separated blastomere <strong>in</strong> zona pellucida prepared from <strong>in</strong>matured oocytes. Manipulated embryos were cultured <strong>in</strong> mCMRL medium until blastocyst stage. By this method, the blastomeres could ma<strong>in</strong>ta<strong>in</strong> cell-to-cell <strong>in</strong>teraction with each blastomere after cleavage, and resulted <strong>in</strong> blastocysts with normal morphology. ICMs were collected from the embryos by immunosurgery, and cultured on mitotically <strong>in</strong>activated-MEF fee<strong>de</strong>r layer. Pluripotency and undifferentiated status of ESCs were evaluated by RT-PCR, immunocytochemical sta<strong>in</strong><strong>in</strong>g and formation of teratoma. Results Two ESCs were established from separated blastomeres (Two-cell; 1/8, Four-cell; 1/4, Normal embryos as control; 3/11). Both cell l<strong>in</strong>es expressed ALP, Oct-4, Nanog, SSEA-4, TRA1-60 and TRA1-81 <strong>in</strong> undifferentiated state. Furthermore, differentiation potencies were also <strong>de</strong>term<strong>in</strong>ed by <strong>in</strong> vitro differentiation and teratoma formation. Conclusion We succeed to establish ESCs from separated blastomeres of non-human primate. It could be important mo<strong>de</strong>l for <strong>de</strong>velopmental competence of cleaved blastomeres, and suggest an effective tool for pre-cl<strong>in</strong>ical research for a regenerative medic<strong>in</strong>e. P530 Effects of physiological and non-physiological <strong>in</strong>sul<strong>in</strong>like growth factor-1 (IGF-1) concentrations on the <strong>in</strong> vitro <strong>de</strong>velopment of bov<strong>in</strong>e embryos Velazquez, MA 1,2 *; Korsawe, K 1 ; Niemann, H 1 1Department of Biotechnology, Institute for Animal Breed<strong>in</strong>g, Mariensee, Neustadt, Germany; 2 Escuela Superior <strong>de</strong> <strong>Ciencias</strong> Agropecuarias, Universidad Autonoma <strong>de</strong> Campeche, Mexico Introduction Physiological concentrations of IGF-1 can exert positive effects <strong>in</strong> bov<strong>in</strong>e embryos. However, <strong>de</strong>trimental effects on bov<strong>in</strong>e oocyte morphology (<strong>Reproduction</strong> 133:1121-28, 2007) and mur<strong>in</strong>e embryo <strong>de</strong>velopment were found with high concentrations of IGF-1, resembl<strong>in</strong>g the non-physiological IGF-1 levels associated with early pregnancy loss <strong>in</strong> women with the polycystic ovary syndrome (Diabetes 56:2228-34, 2007). Determ<strong>in</strong>ation of the effects of normal and abnormal IGF-1 milieus on embryo <strong>de</strong>velopment is important for the un<strong>de</strong>rstand<strong>in</strong>g of embryonic plasticity <strong>in</strong> or<strong>de</strong>r to improve the effectiveness of embryo technologies. The goal of this study was to <strong>de</strong>term<strong>in</strong>e the effects of physiological and non-physiological concentrations of IGF-1 on the <strong>de</strong>velopment and number of cells of <strong>in</strong> vitro-produced bov<strong>in</strong>e embryos. Materials and methods Oocytes obta<strong>in</strong>ed by slic<strong>in</strong>g from abattoir ovaries were matured (24 hrs) and fertilized (18 hrs) <strong>in</strong> vitro. For culture, zygotes were placed randomly <strong>in</strong> three groups: a) control (synthetic oviduct fluid [SOF]/Bov<strong>in</strong>e serum album<strong>in</strong> [BSA]), b) physiological IGF-1 (SOF/BSA + 100 ng/mL IGF-1) or c) nonphysiological IGF-1 (SOF/BSA + 1000 ng/mL IGF-1). On day 8, blastocyst rates were recor<strong>de</strong>d and differential cell sta<strong>in</strong><strong>in</strong>g was applied to count the number of <strong>in</strong>ner cell mass (ICM) and trophecto<strong>de</strong>rm (TE) cells. Data from 14 replicates were analyzed statistically. Results Cleavage was improved by both 1000 (62.7 ± 2.1, P=0.001) and 100 (59.3 ± 3.7, P=0.04) ng IGF-1 over controls (52.4 ± 3.2). The proportion of hatched blastocysts was enhanced by 100 (5.1 ± 1.3, P=0.006) and 1000 (4.1 ± 0.8, P=0.02) ng IGF-1 compared to controls (1.9 ± 0.4). Total blastocyst rate was <strong>in</strong>creased by 100 ng IGF-1 (30.9 ± 1.8, P=0.03) over controls (25.0 ± 1.9), but not by 1000 ng IGF-1 (28.4 ± 2.0 P=0.25). There was a ten<strong>de</strong>ncy (P = 0.07) for the 100 ng IGF-1 group (45.0 ± 4.5) to have less <strong>de</strong>generated embryos compared to 1000 ng IGF-1 (53.9 ± 3.8). 1000 ng IGF-1 <strong>in</strong>creased the number of cells <strong>in</strong> the ICM (37.0 ± 2.2, P=0.007) and the proportion of blastocysts (22.8% P=0.01) with an <strong>in</strong>creased ICM:total cell ratio (≥ 40 %) compared to controls (26.0 ± 1.4 and 0% respectively). Perspective Gene expression and apoptosis analysis are nee<strong>de</strong>d to further characterized the effects of different IGF-1 milieus on bov<strong>in</strong>e preimplantation embryo <strong>de</strong>velopment. M.A.V. is supported by DAAD.
16 t h International Congress on Animal <strong>Reproduction</strong> Poster Abstracts 203 Poster 20 - Gene Modified <strong>Animals</strong> (Transgenics) P531 Glycemia and lept<strong>in</strong>emia are related to changes <strong>in</strong> reproductive fitness of transgenic mice overexpres<strong>in</strong>g pancreatic glucok<strong>in</strong>ase Ballester, J 1 *; Agudo, J 1 ; Bosh, F 1 ; Rodriguez-Gil, JE 2 1CBATEG, Autonomous University of Barcelona, E-08193 Bellaterra. Spa<strong>in</strong>; 2Dept. Animal Medic<strong>in</strong>e and Surgery, Autonomous University of Barcelona, E- 08193 Bellaterra. Spa<strong>in</strong> This study was focused <strong>in</strong> the effects of the overexpression of the glucok<strong>in</strong>ase (GK) gene <strong>in</strong> pancreatic islets on reproductive fitness by utilis<strong>in</strong>g an specific Ins-GK transgenic mice show<strong>in</strong>g this phenotype. The GK overexpression <strong>in</strong>duced a clear drop <strong>in</strong> the prolificacy of Ins- GK mice (8.14±0.83 newborn/<strong>de</strong>livery <strong>in</strong> control mice vs. 3.73±0.17 newborn/<strong>de</strong>livery <strong>in</strong> Ins-GK animals). At the same time, Ins-GK animals showed a clear hypoglycemia (90.4±2.9 mg/dL <strong>in</strong> control mice vs. 61.3±2.4 mg/dL <strong>in</strong> Ins-GK animals) besi<strong>de</strong>s a significant (P