Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 202 Poster Abstracts P528 Development of reconstructed embryos from human somatic cells and ovine enucleated oocytes and isolation of putative human embryonic stem cell clones Zhou, MH*; Zhao, J Faculty of Bioengineering, Inner Mongolia Agricultural University, China Introduction Since the difficulty of obtaining human embryos limited the production of human embryo stem cells this research was to clone heterogeneous embryos by using human fetal skin fibroblast cells as nuclear donor cells and the enucleated ovine oocytes as recipient cytoplasts for examining the developmental potential of the reconstructed embryos to attempt to isolate human embryonic stem cells. Methods Ovine oocytes were collected from slaughterhouse-derived ovaries, in vitro matured and enucleated then used as recipient cytoplasm. Human fibroblast cells was obtained from an aborted 4- month aged fetus skin and employed as donor somatic cells. The cell was injected to sub-zona pellucida of oocyte and fused electrically into ooplasm then activated by Inomycin with 2 M/ml 6- dimethylaminopurine (6-DMAP). Only the activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% (v/v) fetal calf serum (FCS). These heterogeneously cloned embryos were morphologically monitored for their developmental competence at 48h to 168 h of culture. The cell mass of morula or blastocysts were cultured on mouse fibroblast feeder layer and used for the isolation of embryonic stem cell clones. The clones of putative human embryonic stem cell were identified by alkaline phosphatase (AKP). Results The human fetal skin fibroblast cells maintained morphologically normal characteristics and normal numbers of chromosomes (2n = 64) in culture. The reconstructed human embryos completed the first cleavage of between 24 to 48 h and morula/blastocyst development in 72 h to 168 h after activation. 27% of the reconstructed embryos underwent the cleavage while only 15% of the cleaved embryos proceeded to develop 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 staining of the clones showed positive reaction. Conclusions Human fetal fibroblast cell nuclei can be reprogrammed in ovine enucleated oocytes. The heterogeneously nuclear-transferred human embryos can undergo the meiosis division and subsequent development to morula/blastocyst stage thus serving as an alternative for obtaining human embryonic stem cells. The improvement of the reconstructed embryo developmental ability, however, is a future challenge. Poster 19 - Biomedical Models in Reproductive and Regenerative Medicine P529 Embryonic Stem Cells derived from Separated Blastomeres of Cynomolgus Monkey as a Model System of Regenerative Medicine 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 Engineering, Kinki University, Japan; 2 Dept of Orthopedic Surgery, Kinki University, Japan; 3 Keari Co. Ltd., Japan Objective Recently, ESCs (Embryonic Stem Cells) were established from single blastomeres of preimplantation embryos in mice and humans. These methods have been suggested to reduce ethical concerns since we can obtain pluripotent ESCs without interfering embryonic development. Furthermore, these results also give rise to important information about polarity of mammalian embryos. Although totipotencies of separated blastomeres have been experimentally determined in rodents and some domestic animals, only one live-birth was reported in primate. Here, we succeeded in establishment of ESCs from single blastomeres of two-cell and fourcell embryos in cynomolgus monkey. Methods In this study, we cultured the separated blastomere in zona pellucida prepared from inmatured oocytes. Manipulated embryos were cultured in mCMRL medium until blastocyst stage. By this method, the blastomeres could maintain cell-to-cell interaction with each blastomere after cleavage, and resulted in blastocysts with normal morphology. ICMs were collected from the embryos by immunosurgery, and cultured on mitotically inactivated-MEF feeder layer. Pluripotency and undifferentiated status of ESCs were evaluated by RT-PCR, immunocytochemical staining 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 lines expressed ALP, Oct-4, Nanog, SSEA-4, TRA1-60 and TRA1-81 in undifferentiated state. Furthermore, differentiation potencies were also determined by in vitro differentiation and teratoma formation. Conclusion We succeed to establish ESCs from separated blastomeres of non-human primate. It could be important model for developmental competence of cleaved blastomeres, and suggest an effective tool for pre-clinical research for a regenerative medicine. P530 Effects of physiological and non-physiological insulinlike growth factor-1 (IGF-1) concentrations on the in vitro development of bovine embryos Velazquez, MA 1,2 *; Korsawe, K 1 ; Niemann, H 1 1Department of Biotechnology, Institute for Animal Breeding, Mariensee, Neustadt, Germany; 2 Escuela Superior de Ciencias Agropecuarias, Universidad Autonoma de Campeche, Mexico Introduction Physiological concentrations of IGF-1 can exert positive effects in bovine embryos. However, detrimental effects on bovine oocyte morphology (Reproduction 133:1121-28, 2007) and murine embryo development were found with high concentrations of IGF-1, resembling the non-physiological IGF-1 levels associated with early pregnancy loss in women with the polycystic ovary syndrome (Diabetes 56:2228-34, 2007). Determination of the effects of normal and abnormal IGF-1 milieus on embryo development is important for the understanding of embryonic plasticity in order to improve the effectiveness of embryo technologies. The goal of this study was to determine the effects of physiological and non-physiological concentrations of IGF-1 on the development and number of cells of in vitro-produced bovine embryos. Materials and methods Oocytes obtained by slicing from abattoir ovaries were matured (24 hrs) and fertilized (18 hrs) in vitro. For culture, zygotes were placed randomly in three groups: a) control (synthetic oviduct fluid [SOF]/Bovine serum albumin [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 recorded and differential cell staining was applied to count the number of inner cell mass (ICM) and trophectoderm (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 increased 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 tendency (P = 0.07) for the 100 ng IGF-1 group (45.0 ± 4.5) to have less degenerated embryos compared to 1000 ng IGF-1 (53.9 ± 3.8). 1000 ng IGF-1 increased the number of cells in the ICM (37.0 ± 2.2, P=0.007) and the proportion of blastocysts (22.8% P=0.01) with an increased ICM:total cell ratio (≥ 40 %) compared to controls (26.0 ± 1.4 and 0% respectively). Perspective Gene expression and apoptosis analysis are needed to further characterized the effects of different IGF-1 milieus on bovine preimplantation embryo development. M.A.V. is supported by DAAD.
16 t h International Congress on Animal Reproduction Poster Abstracts 203 Poster 20 - Gene Modified Animals (Transgenics) P531 Glycemia and leptinemia are related to changes in reproductive fitness of transgenic mice overexpresing pancreatic glucokinase Ballester, J 1 *; Agudo, J 1 ; Bosh, F 1 ; Rodriguez-Gil, JE 2 1CBATEG, Autonomous University of Barcelona, E-08193 Bellaterra. Spain; 2Dept. Animal Medicine and Surgery, Autonomous University of Barcelona, E- 08193 Bellaterra. Spain This study was focused in the effects of the overexpression of the glucokinase (GK) gene in pancreatic islets on reproductive fitness by utilising an specific Ins-GK transgenic mice showing this phenotype. The GK overexpression induced a clear drop in the prolificacy of Ins- GK mice (8.14±0.83 newborn/delivery in control mice vs. 3.73±0.17 newborn/delivery in Ins-GK animals). At the same time, Ins-GK animals showed a clear hypoglycemia (90.4±2.9 mg/dL in control mice vs. 61.3±2.4 mg/dL in Ins-GK animals) besides a significant (P
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