44 ATTEMPTS TO USE SOMATIC CELLS ISOLATED FROM FROZEN BOVINE SEMEN FOR NUCLEAR TRANSFER

Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals. This is especially important when an animal has died and the only viable genetic material available is frozen semen. In our previous studies, epithelial cells were cultured from fresh ovine and bovine ejaculates and blastocyst stage embryos were produced using these cells (Liu J et al. 2007 Biol. Reprod. special issue, 177; Liu J et al. 2008 Reprod. Fertil. Dev. 20, 102). However, growing cells from frozen semen can be difficult. We hypothesized that nuclei of the somatic cells in frozen semen can be used for nuclear transfer even though the cell membrane and cytoplasm are damaged during the semen freezing process. Electrical fusion or piezo assisted direct injection was applied to introduce nuclei of somatic cells isolated from frozen bovine semen (not cultured) into enucleated bovine oocytes. With electrical fusion, only 5 of the 64 (7.8%) recombined couplets fused, which is lower than our normal fusion rate of approximately 70%. Of the 5 fused embryos, one cleaved and developed to a 4-cell stage embryo. Staining with propidium iodide indicated that less than 10% of somatic cells isolated from frozen bovine semen were viable. These results suggest that it might not be practical to introduce nuclei of the cells in frozen semen into oocytes by fusion due to the high proportions of dead cells. Membranes of the cells in frozen semen were hard and difficult to break by piezo pulses or drawing in and out of the injection pipette. Therefore, whole cell intracytoplasmic injection was applied. After couplet recombination, activation was induced by applying two 0.3 kV cm–1, 55 μs direct-current pulses delivered by an Eppendorf Multiporator (Eppendorf, North America) in activation medium that was composed of 0.28 m Mannitol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mm CaCl2 (Sigma-Aldrich), and 0.1 mm MgSO4 (Sigma-Aldrich), followed by incubation in 10 μg mL–1 cycloheximide (Sigma-Aldrich) and 5 μg mL–1 cytochalasin B (Sigma-Aldrich) for 5 h in a humidified 5% CO2, 5% O2, and 90% N2 gas mixture at 38.5°C. Of the 299 recombined embryos, 82 (27.4%) either cleaved or were fragmented. Of the 82 embryos, 48 were examined and 42 (87.5%) were determined to be fragmented (contained intact donor cells). Low cleavage rates were observed in embryos produced by direct injection (4.5%), but no further embryonic development occurred. Cells cultured from fresh bovine semen were used as positive controls for whole cell intracytoplasmic injection. Of the 74 recombined embryos, 73 (98.6%) cleaved and 4 developed into blastocysts. These results highlight the difficulty of obtaining viable embryos by employing nuclear transfer and somatic cells obtained from frozen semen samples. Additional research is warranted given the potential value of this approach for recovering lost genetics.

62 DIFFERENT EFFECT OF TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF PORCINE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETICALLY ACTIVATED EMBRYOS

Accumulating evidence suggests that trichostatin A (TSA), a histone deacetylase inhibitor, can increase the success rate of somatic cloning. The objective of this study was to investigate the effect of 50 nm TSA treatment on the development of porcine somatic cell nuclear transfer (SCNT) and parthenogenically activated (PA) embryos. Cumulus-oocyte complexes were matured in vitro. The oocytes with the first polar body (PB1) were chosen for SCNT, and the rest with PB1 or good morphology were selected for PA by a single 100-μs direct current pulse of 1.6 kV cm–1, the same parameter as for electrical fusion. GFP transgenic fetal fibroblast cells were used as nuclear donors. Data were analyzed using SPSS (13.0; SPSS, Inc., Chicago, IL, USA) with one-way ANOVA. In Experiment 1, immediately after electrical fusion and activation, the reconstructed embryos were randomly cultured in porcine zygote medium 3 (PZM3) with 10 μg mL–1 cytochalasin B (CB) and 10 μg mL–1 cycloheximide (CHX), with either 0 nm (control) or 50 nm TSA for the first 4 h, before being cultured for another 20 h in PZM3 without CB and CHX. After being washed, the embryos were cultured in PZM3 medium without TSA until Day 6 at 39.0°C, 5% CO2, 5%O2, 90% N2, and 100% humidity. The same experimental design was used for PA embryos concurrently. The results showed that there were no significant differences in blastocyst rates for SCNT or PA between control and TSA groups (23.0 ± 6.1% v. 27.9 ± 6.3%; 21.0 ± 1.0% v. 17.5 ± 3.2%, respectively). Neither were there differences in the cell numbers of blastocysts (38.3 ± 5.7 v. 32.2 ± 3.4; 42.2 ± 3.5 v. 39.0 ± 1.9, respectively). In Experiment 2, TSA treatment was prolonged to either 36 or 40 h. The blastocyst rates of SCNT were increased (7.3 ± 1.2% (0 h), 13.3 ± 2.6% (36 h), and 20.0 ± 3.3% (40 h)), whereas those of PA were decreased (46.7 ± 5.0% (0 h), 27.7 ± 6.5% (36 h), and 30.8 ± 6.3% (40 h)). The cell numbers of blastocysts from either SCNT or PA were also decreased (SCNT: 47.5 ± 3.8, 37.5 ± 2.0, and 37.1 ± 3.3; PA: 46.1 ± 1.9, 37.5 ± 1.9, and 39.3 ± 2.2; P < 0.05). In Experiment 3, the cell number and the apoptotic index of Day 5, 6, and 7 PA blastocysts treated with 0 or 50 nm TSA were determined by the terminal deoxynucleotide-mediated nick end labeling (TUNEL) assay (Table 1). The results suggested that TSA treatment probably delayed embryo development, which may be one of the reasons for the lower cell numbers in the TSA-treated group. Table 1. Cell apoptosis of PA blastocyst by TUNEL

374 PRODUCTION OF TRANSGENIC CLONED PIGLETS BY USING PORCINE FETAL FIBROBLASTS OVEREXPRESSING ENDOTHELIAL NITRIC OXIDE SYNTHASE

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by endothelial nitric oxide synthase (eNOS). In addition, eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolism, and cardiovascular systems of pigs and humans. The main goal of this study was to produce pigs that carry an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag so that a better understanding of cardiovascular regulation by eNOS can be gained. Nuclear transfer was conducted to create these animals, and the effects of two different oocyte activation treatments and two different culture systems were examined. Mature oocytes were purchased from BoMed, Inc. (Maidson, WI, USA) and enucleated by micromanipulation. Donor cells were injected into the perivitelline space and electrically fused to the recipient oocyte. Treatment 1 consisted of electrical fusion/activation in 1 mM calcium in mannitol; Treatment 2 consisted of electrical fusion in 0.1 mM calcium in mannitol, followed by chemical activation in 200 �M thimerosal for 10 min followed by 8 mM DTT for 30 min. The fusion rate (71% vs. 82%) was lower (P < 0.0001) in Treatment 2 than in Treatment 1. For those oocytes that fused, the cleavage rates (5.9% vs. 0.4%) at 14-18 h were higher (P < 0.0001) for Treatment 2. Fused oocytes were cultured for 12-18 h in 5% O2, 5% CO2, 90% N2 (low oxygen) or 5% CO2 in air (normal oxygen), and were then surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. In February four cloned transgenic piglets were born (birth weights were 495, 595, 965, and 685 g). Two were from Treatment 1 and low oxygen, and the other two were from Treatment 2 and normal oxygen, and all are currently healthy. PCR, RT-PCR, and Western blotting analysis confirmed that the pigs were transgenic, made message, and made the fusion protein. Immunohistochemistry confirmed that the fusion protein localized in the endothelial cells of the placental vasculature from the conceptuses as did the endogenous eNOS. In addition to the transgenic males, four wild-type females were born (birth weights were 585 (this one died on Day 4) 445, 930, and 585 g). All four were from Treatment 2 and low oxygen, and three are healthy. In conclusion, viable piglets were produced from both Treatments 1 and 2, as well as in both high and low oxygen conditions. Thus, both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system. This work was funded by a grant from the NIH RR18276 and Food for the 21st Century.