bluuebunny/crossref_metadata_2025 huggingface dataset:
285 VIABILITY AND GENE EXPRESSION OF MESENCHYMAL STEM CELLS CRYOPRESERVED WITH DIFFERENT CRYOPROTECTANTS
Authors: Kim M. K. , Ock S. A. , Jeon B. G. , Cho J. H. , Rho G. J.
Published: January 2008

Long-term storage of stem cells with self-renewal plays a pivotal role in cell tissue engineering, which could be a novel option for improving regenerative diseases. However, the choice of a selective cryoprotectant is still to be addressed. Dimethyl sulfoxide (DMSO), which in current practice is the most widely used as a cryoprotectant for cell freezing, is known to have toxic side effects (Wang et al. 2007 Cryobiology 55, 60–65). In this study, therefore, the effect of two different cryoprotectants, used alone or in combination, on frozen–thawed porcine mesenchymal stem cells (MSCs) was investigated by evaluating their viability, apoptosis, and gene expression patterns. MSCs isolated from bone marrow were cultured in advanced Dulbecco's modified Eagle medium (ADMEM) supplemented with 10% fetal bovine serum (FBS) and characterized by the expression of alkaline phosphatase (AP) activity and cell-surface antigen profiles (CD105 as positive, CD45 and CD133 as negative markers). Subconfluent cultures of MSCs (1 � 106 cells mL–1) at 4–5 passages were equilibrated in different cryoprotectants: (1)ADMEM supplemented with 10% DMSO, (2) ADMEM supplemented with 1.5 m ethylene glycol (EG), and (3) ADMEM supplemented with 0.75 m EG and 5% DMSO, and frozen in a programmable freezer. The straws were cooled at –4�C min–1 from 25�C to –7�C. After being seeded, the straws were further cooled to –35�C at a –0.6�C min–1 ramp rate, and then immediately plunged into LN2 and stored at least a week. After thawing at 37�C in a water bath, viability and apoptosis of cells were analyzed by flow cytometry using an In Situ Cell Death Detection kit (Roche, Mannheim, Germany). Expression of HSP70, Nanog, and β-actin was analyzed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), and compared to that of non-cryopreserved MSCs. Doubling time of cryopreserved MSCs was higher than for non-cryopreserved MSCs. There were no significant differences among cryopreserved groups. The rates of viability and apoptosis of non-cryopreserved MSCs (91% and 4.71%, respectively) was significantly (P < 0.05) higher than for MSCs cryopreserved with 10% DMSO, 1.5 m EG, or the 0.75 m EG and 5% DMSO mixture (71.78% and 3.45%, 70.09% and 3.22%, 68.97% and 2.42%, respectively), but the rates among different cryopreserved treatments did not differ. After thawing, expression of HSP70, Nanog, and β-actin in cryopreserved MSCs showed patterns similar to those of non-cryopreserved MSCs. In conclusion, the present study indicates that EG is an alternative cryoprotectant for cryopreservation of porcine MSCs. Further studies are needed to evaluate the possible effects on the expression of genes related to viability and apoptosis in MSCs cryopreserved with different cryoprotectants.
287 ACTIVE MITOCHONDRIA ARE PRESENT IN MOUSE AND MONKEY EMBRYONIC STEM CELL LINES
Authors: Lonergan T. , Harvey A. , Zhao J. , Bavister B. , Brenner C.
Published: January 2008

The inner cell mass (ICM) of the blastocyst develops into the fetus after uterine implantation. Prior to implantation, ICM cells synthesize ATP by glycolytic reactions. We now report that cells of the ICM in 3.5-day-old mouse embryos have too few mitochondria to be visualized with either Mitotracker red (active mitochondria) or an antibody against complex I of OXPHOS. By comparison, all of the surrounding trophectoderm cells reveal numerous mitochondria throughout their cytoplasm. It has largely been assumed that embryonic stem (ES) stem cells derived from the ICM also have few mitochondria, and that replication of mitochondria in the ES cells does not begin until they commence differentiation. We further report that mouse E14 ES cells and monkey ORMES 7 ES cells have considerable numbers of active mitochondria when cultured under standard conditions, i.e., 5% CO2 in air. Both the mouse E14 and monkey ES cell lines expressed two markers of undifferentiated cells, Oct-4 and SSEA-4, and monkey ES cells expressed the undifferentiated cell marker Nanog; however, Oct-4 is nonspecific in monkey ES cells because trophectoderm also expresses this marker, unlike in mice. Ninety-nine percent of the E14 cells examined, and 100% of the ORMES 7 cells, have a visible mitochondrial mass when stained with either Mitoracker red or with an antibody against OXPHOS complex I. The ATP content in the mouse E14 cells (4.13 pmoles ATP/cell) is not significantly different (P = 0.76) from that in a mouse fibroblast control (3.75 pmoles ATP/cell). Cells of the monkey ORMES 7 cell line had 61% of the ATP/cell content (7.55 pmoles ATP/cell) compared to the monkey fibroblast control (12.38 pmoles ATP/cell). Both cell lines expressed two proteins believed to indicate competence of mitochondria to replicate: PolG, the polymerase used to replicate the mitochondrial genome, and TFAM, a nuclear-encoded transcription factor reported to regulate several aspects of mitochondrial function. Both proteins were found to co-localize in the mitochondria. We conclude that when the ICMs are isolated from blastocysts and used to establish these two ES cell lines in cell culture, mitochondrial biosynthesis is activated.
290 A CYNOMOLGUS MONKEY EMBRYONIC STEM CELL LINE DERIVED FROM A SINGLE BLASTOMERE
Authors: Okahara-Narita J. , Yamasaki J. , Iwatani C. , Tsuchiya H. , Wakimoto K. , Kondo Y. , Wakayama T. , Torii R.
Published: January 2008

The establishment of most embryonic stem (ES) cell lines requires the destruction of embryos. Some ES cell lines in mice and humans are currently derived from a single blastomere, so that remaining blastomeres can still develop into fetuses. However, the procedures currently in use for establishing these lines are very complicated, and other ES cell lines from the same species are needed (Chung et al. 2006 Nature 439, 216–219; Klimanskaya et al. 2006 Nature 444, 481–485). The objective of this study was to devise a method simpler than those previously described for establishing ES cell lines from a single blastomere in the cynomolgus monkey. Controlled ovarian stimulation and oocyte recovery have been described previously by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI), and then cultured at 38�C in 5% CO2, 5% O2 for 2 days. The zona pellucida of 4- to 5-cell-stage embryos was disrupted using acidic Tyrode's solution, and individual blastomeres were separated from the denuded embryos using trypsin. These blastomeres were cultured on mitomycin-C-treated mouse embryonic fibroblasts and ES medium containing adrenocorticotropic hormone (ACTH) (Ogawa et al. 2004 Genes to Cells 9, 471–477). After the formation of initial outgrowths, half of the medium was changed every other day until the outgrowths reached approximately 100 cells. Passage of putative monkey ES cells was performed by mechanical dispersion of the colonies and transfer to fresh feeders every 3–4 days until there were enough cells for enzymatic dispersion. One stable ES cell line was obtained from two 4- or 5-cell-stage embryos using ES medium containing ACTH. The morphology of this ES cell colony was consistent with the monkey ES cell colony previous described by Suemori et al. (2001 Dev. Dynamics 222, 273–279). The ES cell line was passaged more than 17 times, and the morphology of the ES cell colony did not differ between the first and seventeenth passages. The ES cells showed normal karyotype and retained pluripotency markers for primate ES cells including octamer-binding protein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-60, and TRA-1-81. We are presently confirming whether this ES cell line possesses potencies to differentiate in all three embryonic germ layers using both an in vitro assay and teratoma formation. Here we showed that cynomolgus monkey ES cells can be derived from a single blastomere, without co-culturing another ES cell line, as has been done in previous studies on mice and humans. This method allows the establishment of ES cell lines from a single blastomere, leaving the other blastomeres available for embryo transfer. Thus, the method described here is simpler than previously described methods and alleviates some ethical concerns.
292 MULTILINEAGE POTENTIAL OF CANINE MESENCHYMAL STEM CELLS DERIVED FROM BONE MARROW
Authors: Song H. J. , Kang E. J. , Jeon B. G. , Rho G. J.
Published: January 2008

Mesenchymal stem cells (MSCs) isolated from bone marrow have unique self-renewal capability for multilineage differentiation and show promise in the field of medicine for cell therapies and tissue engineering. In the present study, we examined the maintenance of cell populations and the differentiation ability of canine MSCs into mesodermal lineages, oesteoblasts and adipocytes, under different culture conditions. MSCs isolated from bone marrow by Ficoll gradient treatment were cultured in (1) ADMEM supplemented with 10% fetal bovine serum (FBS) (basic medium), (2) basic medium supplemented with 10 ng mL–1 basic fibroblast growth factor (bFGF), (3) basic medium supplemented with 1000 IU mL–1 human leukemia inhibitory factor (hLIF), and (4) basic medium supplemented with 10 ng mL–1 bFGF and 1000 IU LIF. In Experiment 1, under a phasecontrast microscope (40�), morphological changes of MSC populations, including the appearance of more spherical shapes and extending processes arranged into network-like structures, were observed in all media with cytokines added, but not in basic medium. In Experiment 2, MSCs cultured under different conditions at 3–5 passages were induced into osteogenic and adipogenic lineages, and their characteristics were evaluated based on the formation of mineral nodules by alkaline phosphatase-positive cells, von Kossa and alizarin red staining of osteoblasts, and oil red O staining of lipid vacuoles following the protocol described earlier (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90). Only MSCs cultured in basic medium were successfully differentiated into osteoblasts and adipocytes. In conclusion, this study indicates that supplementation of culture media with cytokines might have a negative effect on the pluripotency maintenance of canine MSCs. Further studies are required to evaluate more characteristics of gene expression in canine MSCs.
300 HEMIZYGOUS PRION PROTEIN GENE (PRNP) KNOCKOUT IN CATTLE FIBROBLASTS
Authors: Brunetti D. , Rossi G. , Lagutina I. , Duchi R. , Colleoni S. , Catania M. , Viscomi C. , Piga D. , Zeviani M. , Lazzari G. , Tagliavini F. , Galli C.
Published: January 2008

Bovine spongiform encephalopathy (BSE) represents a real threat for human health, as has been demonstrated by the causal link with the variant form of Creutzfeldt-Jakob disease. The aim of our project is to create a bovine strain knockout for the prion protein gene (PRNP) that should be resistant to BSE infection. We combined the use of homologous recombination by PRNP targeting vectors in bovine fibroblasts with the subsequent use of nuclear transfer (NT). We transfected fetal (male) and adult (female) bovine fibroblasts by nucleofection, using targeting vectors disrupting the PRNP by means of loxP flanked cassettes. They expressed resistance to different drugs driven by a PGK or TK promoter and the thymidine kinase gene as a negative selection marker. We screened, by PCR, 907 drug-resistant colonies, from which we identified 8 Neo-resistant colonies with a recombined PRNP allele (overall efficiency 3.2%; 7/108 from fetal, 1/145 from adult; P < 0.5). Fibroblasts PRNP+/– Neo were used to produce NT blastocysts from which neural precursors cell lines were established (Lazzari et al. 2006 Stem Cells 24, 2514–2521). These lines were capable of extensive proliferation (over 120 doublings during 4 months of culture) and provided unlimited material for Southern blot analysis to confirm PCR findings. Three clones (2 from fetal and 1 from adult) were further analyzed and confirmed PRNP+/– by Southern blot and were subsequently used for NT to generate blastocysts for transfer to recipient heifers. On Day +40 of gestation, the pregnancy rate was 33.3% (9/30) for the fetal line and 50% (2/4) for the adult line. One of the fetuses originating from fetal fibroblasts was removed on Day +45 to establish a rejuvenated fibroblast cell line used for a second round of gene targeting to obtain a PRNP –/– clone. We nucleofected these fibroblasts with Puro, Hygro, and promoterless Hygro cassette-carrying targeting vectors. We screened 625 drug-resistant colonies by PCR but none tested positive for the second targeting. In conclusion, we have obtained heterozygous PRNP+/– fibroblasts with the Neo vector both in fetal and adult fibroblasts, but failed with other vectors. In the first targeting, the efficiency was 10 times greater in fetal v. adult fibroblasts. The derivation of neural precursor cell lines from cloned blastocysts is a useful procedure to have sufficient material for molecular analysis without the need of rejuvenating the cell through the production of a fetus. None of the vectors used for the targeting of the second allele was successful.
302 INHIBITION OF HUMAN NATURAL KILLER (NK) CELL-MEDIATED CYTOTOXICITY IN MINIPIG CELLS BY KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS K5 PROTEIN
Authors: Han G. S. , Choi K. M. , Hong S. P. , Yoo J. Y. , Kim E. J. , Kim S. H. , Park Y. C. , Seol J. G. , Park K. W.
Published: January 2008

Human natural killer (NK) cell-mediated response plays an important role in xenograft rejection. In the case of pig-to-human xenotransplantation, it has been suggested that NK cells are involved in delayed-type rejection, which is characterized by pig endothelial cell activation, direct lysis, and secretion of proinflammatory cytokines. Natural killer cell activation can be a direct barrier to the potential use of pig organs for human xenograft transplantation. Therefore, it is important to suppress NK cell activity on pig-to-human xenografts. Expression of Kaposi's sarcoma-associated herpesvirus (KSHV)-K5 molecules inhibits the cytotoxic activity of NK-activating receptor (B7-2, ICAM-1). As a consequence, K5 expression drastically inhibits NK cell-mediated cytotoxicity. In this study, we produced cell lines expressing K5 to control NK-mediated cytotoxicity in minipig cells. We transfected the K5 gene into minipig fetal fibroblasts and established 2 transgenic clonal cell lines. Presence of the K5 gene was confirmed by PCR, and expression of the gene was identified by real-time PCR and flow cytometry. In an NK cytotoxicity assay, the rate of NK-92MI-mediated cytotoxicity was significantly reduced, to 48.4 � 5.9% compared with the control (75.6 � 5.8%; P < 0.05, n = 8, paired t-testing). In conclusion, these results indicate that the expression of K5 molecules on porcine cells can efficiently control NK-mediated cytotoxicity. This strategy can be used in transgenic pig production in which porcine organs would be protected from NK-mediated rejection.
315 CORPORA LUTEA MORPHOLOGICAL AND ECHOTEXTURAL ATTRIBUTES RELATED TO PLASMA PROGESTERONE CONCENTRATION IN HEIFERS
Authors: Siqueira L. G. B. , Viana J. H. M. , Torres C. A. A. , Souza E. D. , Amorim L. S. , Fonseca J. F. , Camargo L. S. A. , Monteiro Jr P. L. J.
Published: January 2008

It has been suggested that ultrasound image attributes are a potential indicator of the physiological and functional status of the corpus luteum (CL). The aim of this study was to evaluate corpus luteum morphological and echotextural changes, and to correlate these changes with plasma progesterone concentration [P4] throughout the bovine estrous cycle. Crossbred heifers were scanned daily, using a B-mode, real-time ultrasound machine equipped with a 5-MHz linear-array rectal transducer, throughout a natural estrous cycle (Experiment 1; n = 12) or during a shorter estrous cycle, interrupted on the 10th day, by luteolysis induction (Experiment 2; n = 6). Blood samples were collected for further plasma [P4] analyses by RIA. Corpora lutea areas (cm2) were measured, and daily images of each CL were videotaped (VHS tapes) until digitized. Computer-assisted analyses of image attributes were performed using a custom-developed software. Daily values of luteal area, echotexture, and plasma [P4] values were analyzed by ANOVA with Tukey's test to determine differences among means of each cycle day. Pearson's correlation coefficients were calculated between luteal area, mean pixel value, pixel heterogeneity, and plasma [P4]. In the first experiment, luteal tissue area increased to a maximum on the 10th day (P < 0.05), followed by a plateau, and then declined from Day 14 to next estrus. There was a significant correlation between luteal tissue area and plasma P4 (r = 0.69; P < 0.01). In the second experiment, plasma P4 dropped to basal values 24 h after luteolysis induction. Luteal tissue area decreased at a slow rate, and reached values similar to ones from metestrus 36 h after treatment. In Experiment 1, echotexture parameters of the CL were analyzed after data adjustment to the onset of luteolysis. In both experiments, mean pixel values did not change throughout the estrous cycle and there was no correlation between mean pixel values and plasma [P4] (P > 0.10). Pixel heterogeneity changed throughout the natural estrous cycle, with maximum value on metestrus (Day 14; Day 0 = luteolysis) and minimum on diestrus (Day 2; P < 0.01). However, this parameter did not change when luteolysis was induced (Experiment 2; P > 0.10). There were significant correlations between pixel heterogeneity and plasma progesterone in both of the experiments (r = –0.69 and r = –0.48; P < 0.05). In conclusion, mean pixel values do not reflect morphological or functional changes of the CL throughout the estrous cycle. On the other hand, based on the correlations between pixel heterogeneity and systemic [P4] in both experiments, this image attribute (heterogeneity) has the potential to indicate functionality and steroidogenic capacity of the luteal gland.
33 PRE-AND POST-IMPLANTATION DEVELOPMENT OF EMBRYOS CLONED FROM PORCINE SKIN-DERIVED SPHERE STEM CELLS
Authors: Hao Y. H. , Wax D. , Zhong Z. S. , Murphy C. N. , Spate L. , Samuel M. , Rieke A. , Dyce P. , Li J. L. , Prather R. S.
Published: January 2008

Although transgenic animals have been successfully cloned, the process is still inefficient. One of the limitations is the use of somatic donor cells that have a limited lifespan. If a genetic modification is made, the selection process must be initiated and completed rapidly or the cells will undergo senescence. Identification of a stem cell that would proliferate rapidly and not undergo senescence would prove to be very valuable. Here we report attempts at cloning by using porcine skin-derived sphere stem cells to determine if they are a suitable donor cell type. Skin-derived stem cells were isolated from fetal skin and express the neural progenitor marker NES, as well as genes that may be critical for pluripotency such as POU5F1 and STAT3. The skin-derived stem cells proliferate rapidly in vitro and retain a normal karyotype after long-term culture. In the present study, skin-derived stem cells were cultured and frozen in liquid nitrogen from passage 1 to passage 8. To investigate the developmental potential of the skin-derived stem cells, we performed nuclear transfer (NT) and compared their preimplantation developmental efficiency to that of the embryos derived from in vitro fertilization (IVF). Cumulus–oocyte complexes (COCs) were aspirated from antral follicles of ovaries from prepubertal gilts. Approximately, groups of 50-70 COCs were matured in vitro in 500 µL TCM-199 per culture well for 40–44 h at 38.5�C, in a humidified atmosphere of 5% CO2 in air. The donor cells were thawed and cultured one day before NT; skin-derived stem cells were pipetted vigorously in PBS-EDTA to isolate individual cells. For IVF, cryopreserved ejaculated spermatozoa were thawed and washed and then resuspended with fertilization medium (mTBM). The MII oocytes were co-incubated with sperm for 6 h, and then transferred to PZM3 and cultured. For NT and IVF, respectively, the percent cleavage at 48 h in PZM3 was 64.9 � 8.2% (169/208) and 62.1 � 3.1% (94/184) (P > 0.05), the percent blastocysts after 6 days was 21.5 � 5.8% (53/208) and 25.2 � 3.4% (46/184) (P > 0.05), and the number of nuclei per blastocyst was 28.5 � 1.9 (NT, maximum was 58) and 16.8 � 4.0 (IVF, maximum was 31) (P < 0.05). To determine development post-implantation, some cloned embryos were cultured in PZM3 for 15.5 h and an average of 112 cloned embryos were transferred to the oviducts of four naturally cycling gilts on Day 0–1 of standing estrus. Three of the animals were pregnant: one of them farrowed two male piglets on August 14th, with the other two due on September 8th and 9th. Future studies will involve performing NT and ET on skin-derived stem cells from a higher passage number to determine if they would be suitable for genetic modification prior to NT.
35 IN VITRO DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS DERIVED FROM PORCINE EMBRYONIC GERM CELLS AND THEIR DESCENDENT NEURAL PRECURSOR CELLS
Authors: Heo S. Y. , Ahn K. S. , Na D. C. , Won J. Y. , Seok H. B. , Choi S. J. , Shim H.
Published: January 2008

Due to an ease of reprogramming in the process of nuclear transfer (NT), undifferentiated stem cells may support greater development of cloned embryos compared with differentiated cell types. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than somatic cells. In pigs, comparative studies of NT using stem cells and their differentiated descendant cells have not yet been reported. Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated. Porcine EG cells were isolated from cultured PGC collected from embryos on Day 23 of gestation (Shim et al. 1997 Biol. Reprod. 57, 1089–1095). Neural precursor cells, based on morphology of neural rosettes or neurospheres, were differentiated from porcine EG cells by sequential culture of the cells on gelatinized dishes in N2B27 medium with and without 10 µm retinoic acid for 7 days each. Since porcine EG cells and NP cells differentiated from the same EG cells were used in the present study, the genetic makeup of nuclear donor cells was identical, eliminating any genetic variation between undifferentiated and differentiated cells in the comparison. The efficiencies of NT using the two different cell populations were compared. The rates of fusion were not different between NT embryos from EG and NP cells. However, the rate of cleavage in NT embryos from EG cells was significantly higher (P < 0.05) than that from NP cells (141/247, 57.1% v. 105/228, 46.1%). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than that using NP cells (43/247, 17.4% v. 18/228, 7.9%). The effects on NT efficiency using either stem cells or their differentiated progeny is still controversial. The rates of blastocyst development using somatic stem cells were either reduced in mice (Sung et al. 2006 Nat. Genet. 38, 1323–1328) or remained the same in deer (Berg et al. 2007 Biol. Reprod. 77, 384–394) and cattle (Green et al. 2007 Biol. Reprod. 77, 395–406). However, the results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. This suggests that the effect on NT efficiency by using stem cells and their differentiated progeny may vary depending upon the species and cell types involved.
37 EFFECTS OF TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Iager A. E. , Beyhan Z. , Ross P. J. , Ragina N. P. , Cunniff K. , Rodriguez R. M. , Cibelli J. B.
Published: January 2008

Faulty epigenetic reprogramming is a likely major cause of the low success rate observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been reported that treatment of reconstructed mouse embryos with the potent histone deacetylase inhibitor, trichostatin A (TSA), results in significantly increased developmental capacity of SCNT preimplantation embryos and live offspring (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 240, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089; Kishigami et al. 2006 J. Reprod. Dev. 53, 165–170). Studies investigating similar reprogramming capabilities of TSA in bovine SCNT embryos report conflicting results (Akagi et al. 2007 Reprod. Fertil. Dev. 19, 24 abst; Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 48 abst). In this study, the effects of TSA treatment on in vitro development of bovine SCNT embryos were examined. Bovine fetal fibroblasts were cultured under contact inhibition for 2 to 5 days and used as donor cells for SCNT. Oocytes were aspirated from abattoir-derived ovaries, and matured in vitro for 18 h prior to enucleation. Reconstructed SCNT couplets were electrofused, and then activated 24 h post-maturation using 5 µm ionomycin followed by 2 mm dimethylaminopurine (DMAP) for 4 h. SCNT embryos were subjected to 0 (control; C-NT) or 50 nm TSA for 13 h post-ionomycin (hpi) TSAa-NT) or 13 hpi + 6 h starting from 40 hpi (TSAb-NT). IVF embryos were produced as an additional control. All embryos were cultured in KSOM supplemented with 3 mg mL–1 BSA for 7.5 days, with 5% FBS added on Day 3. Experiments were repeated 3 or 7 times, and data were analyzed a -way ANOVA procedure. Developmental rates to the blastocyst stage and total cell number of blastocysts were determined. Total cell numbers were determined by fixing blastocysts in 4% paraformaldehyde, and staining with bisbenzimide 33342, followed by microslide mounting and visualization using an epifluorescence microscope. No difference was observed in cleavage rates among the four treatment groups, C-NT, TSAa-NT, TSAb-NT, and IVF, with the rates being 66%, 75%, 73.1%, and 82.3%, respectively (P = 0.33); nor was any improvement seen in the rate of blastocyst development of TSAa-NT or TSAb-NT over C-NT embryos: 36%, 40.2%, and 30.2%, respectively (P = 0.22). Furthermore, there was no significant difference in mean total cell number of blastocysts among treatment groups: C-NT, 120.2; TSAa-NT, 124.2; TSAb-NT, 129.3; and IVF, 141.1 (P = 0.29). These results suggest that 50 nm TSA treatment immediately following activation does not affect the development of bovine SCNT preimplantation embryos.
38 EFFECTS OF TRICHOSTATIN A ON DNA METHYLATION IN CLONED BOVINE EMBRYOS
Authors: Iwamoto D. , Kishigami S. , Taniguchi S. , Abe Y. , Matsui T. , Kasamatsu A. , Tatemizo A. , Mitani T. , Kato H. , Matsumoto K. , Hosoi Y. , Wakayama T. , Iritani A. , Saeki K.
Published: January 2008

Recently, the efficiency of full-term development of somatic cloned mouse embryos was significantly increased by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). We have shown that TSA treatment improved the rate of development of the cloned bovine embryos to the blastocyst stage (Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 142 abst). Higher levels of DNA methylation have been shown in early cloned bovine embryos than in in vitro-fertilized (IVF) embryos (Dean et al. 2001 Proc. Nat. Acad. Sci. USA 98, 13734–13738; Santos et al. Curr. Biol. 13, 1116–1121). In this study, we examined the effects of TSA on DNA methylation levels in cloned bovine embryos by immunostaining with an antibody to 5-methyl cytosine (5-MeC). Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days before they were used as donor cells. The cells were electrofused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. Atotal of 131 cloned embryos were produced. The NT embryos were exposed to 0 (control) and 50 nmTSA from the start of activation to 48 h post-activation (hpa). They were then cultured in an mSOF medium. At 60 hpa, only embryos developed to the 8-cell stage were used for assessment of DNA methylation levels. Sixteen TSA-treated, 22 non-treated, and 19 IVF embryos were immunostained with 5-MeC antibody. For quantitative analysis of the DNA methylation levels, 5-MeC signals in the fluorescent images were determined using an image analyzer system (Aqua Cosmos; Hamamatsu Photonics, Shizuoka, Japan). The data were analyzed with Tukey-Kramer post hoc test for multiple comparisons following ANOVA. Relative levels of DNA methylation of TSA-treated cloned and IVF embryos did not differ (P > 0.05), but were lower than those of non-treated cloned embryos (P < 0.05). The results indicate that TSA treatment of cloned bovine embryos leads to a reduction of DNA methylation levels of their genome. The data suggest that the TSA treatment decreased the DNA methylation levels of cloned bovine embryos to the levels of IVF embryos, resulting in improved blastocyst development of the cloned embryos.
49 EARLY ASPECTS OF NUCLEAR REPROGRAMMING FOLLOWING BOVINE SOMATIC CELL NUCLEAR TRANSFER
Authors: Petrovicova I. , Svarcova O. , Strejcek F. , Lucas-Hahn A. , Lemme E. , Niemann H. , Laurincik J. , Maddox-Hyttel P.
Published: January 2008

This study was designed in order to evaluate the global transcription and morphology of reprogramming during somatic cell nuclear transfer (SCNT). Following 20–22 h of IVM, couplets of MII cytoplasts and starved bovine fibroblasts were, after 2 h co-culture, electrically fused, chemically activated by 5 µm ionomycin for 5 min, followed by a 3–4 h incubation in 2 mm 6-DMAP, and fixed at 0.5, 1, 1.5, 2, 3, 4, 8, 12, and 16 h post-activation (hpa). The reconstructed embryos were processed for lacmoid staining, autoradiography following [3H]uridine incubation, transmission electron microscopy (TEM), and immunofluorescence (4 and 12 hpa) in order to evaluate chromatin dynamics, transcriptional activity, nuclear and nucleolar ultrastructure, and localization of nucleolar proteins (upstream binding factor (UBF) and fibrillarin), respectively, during the first cell cycle. Likewise, starved fibroblasts were fixed and processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillo-granular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. During first 3 hpa, the majority of the embryos displayed a single block of condensed chromatin surrounded by a more or less complete nuclear envelope (NE) and an abundant population of elongated somatic cell mitochondria. This somatic cell complex was located peripherally in the ooplasm. At the subsequent time points, the embryos displayed pronuclear-like structures, which from 8 hpa were located centrally in the ooplasm. From about 4 hpa, the somatic cell complex had dispersed and the elongated mitochondria could no longer be tracked. The first nucleolus-related structures were observed at 1.5 hpa and only in nuclei with a complete NE. At 1.5 to 4 hpa, the nucleolus-related structures appeared either as bodies presenting a large fibrillar center and presumptive dense fibrillar component, but no granular component, or as electron-dense nucleolus precursor bodies (NPBs). From 4 hpa and onward, only compact NPBs were observed. At 4 and 12 hpa, UBF was localized into small discrete clusters of foci enclosed in a shell-like structure labeled by fibrillarin in the nucleus. In conclusion, at SCNT, the somatic cell cytoplasm remains structurally organized in a somatic cell complex over the initial 3–4 hpa. During the same period, the somatic cell chromatin undergoes condensation and the NE is partially dissolved. Subsequently, pronucleus-like euchromatic nuclei with typical NPBs are formed, although somatic cell nucleolar components may be temporarily seen. Throughout the process, transcription is repressed.
This work was supported by Marie Curie Intra-European Fellowships (MEIF-CT-2006-021629), and by grant VEGA 1/3255/06 and DFG.
60 INFLUENCE OF HOECHST STAINING FOR NUCLEAR TRANSFER ON PARTHENOGENETIC EMBRYOS IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS)
Authors: Tsuchiya H. , Iwatani C. , Okahara-Narita J. , Yamasaki J. , Torii R.
Published: January 2008

Nonhuman primates are valuable animal models for the study of human diseases, and somatic cell nuclear transfer (SCNT) is an important method for establishing tailor-made embryonic stem (ES) cells and transgenic animals in these model species. However, there have been few reports on SCNT in nonhuman primates. Moreover, the development of cloned blastocysts could be influenced by any chemical reagents and manipulations used in this technique. In this study we compared blastocyst developmental rates with and without Hoechst staining. Metaphase II (MII) oocytes were collected from hormone-treated adult female cynomolgus monkeys (Macaca fascicularis) under laparoscopic observation (Torii et al. 2000 Primates 41, 39–47). A pseudo-SCNT procedure, which consisted of cytochalasin B treatment, cytoplasm removal, and dissection of the oocyte membrane, was performed on MII oocytes either in the presence of (Experiment 1; Ex1) or in the absence of Hoechst 33342 (Experiment 2; Ex2). Hoffman modulation contrast microscopy was used in Ex1 and Nomarski differential interference contrast (DIC) was used in Ex2. In Ex1, cumulus-free MII oocytes were treated with Hoechst 33342 (5 mg mL–1; Sigma Chemical Co., St. Louis, MO, USA) for 5 min and the following pseudo-SCNT procedure was carried out: cytochalasin B (CB, 5 µg mL–1; Sigma) for 20 min, removal of a small amount of cytoplasm (pseudo-EN), and then dissection of the oocyte cytoplasmic membrane (pseudo-IN) under Hoffman modulation contrast microscopy. In Ex2, CB treatment, pseudo-EN, and pseudo-IN were performed under Nomarski DIC microscopy. After treatment, these oocytes were activated by parthenogenetic stimulation. Parthenogenesis was induced by 5-m ionomycin (Sigma) for 2 min and 2 mm 6-dimethylaminopurine (Sigma) for 4 h. As a control, cumulus-free MII oocytes were activated by only parthenogenetic stimulation, without the above manipulations. These activated oocytes were cultured in CMRL-1066 medium containing 20% calf serum at 38�C in 5% CO2, 5% O2, and 90% N2 for 7–8 days. The rates of development to blastocyst stage were 14% (1/7) in Ex1, 30% (3/10) in Ex2, and 29% (2/7) in the control. The developmental rate of parthenotes to the blastocyst stage in Ex2 was greater than that in Ex1 and similar to the control. These results suggest that treatment of cynomolgus monkey oocytes with Hoechst staining possibily decreases development to the blastocyst stage. Therefore, enucleation under Nomarski DIC will be a good alternative to Hoechst staining and could improve the potential development of nonhuman primate SCNT embryos.
62 DIFFERENT EFFECT OF TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF PORCINE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETICALLY ACTIVATED EMBRYOS
Authors: Wei H. X. , Zhang K. , Ma Y. F. , Li Y. , Li Q. Y. , Dai Y. P. , Li N.
Published: January 2008

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
69 REMOVAL OF CUMULUS CELLS AND PRE-INCUBATION IN CYTOCHALASIN B IMPROVE SURVIVAL OF IMMATURE CAT OOCYTES DURING VITRIFICATION
Authors: Comizzoli P. , Wildt D. E. , Pukazhenthi B. S.
Published: January 2008

Vitrification might be the best option to cryopreserve the immature cat oocyte because our recent studies have demonstrated that COCs are (1) highly sensitive to cryoprotectant (CPA) and require short-term exposure to prevent toxicity, and yet (2) resistant to extreme hyperosmotic conditions when pre-incubated in the cytoskeleton stabilizer cytochalasin B (CB). However, surrounding cumulus cells may inhibit the necessary rapid transport of water and CPA across the oocyte membrane during short-term exposure to vitrification solutions (VS) containing high CPA concentrations. The objective was to examine the influence of cumulus cells on oocyte survival during vitrification with or without pre-incubation in CB. Our metric of focus was the chromatin status after warming and IVM. Grade I immature oocytes (n = 420, 4 replicates) were equally allocated to one of four treatments: T1 (removal of cumulus cells in 0.2% hyaluronidase); T2 (pre-incubation in 7.5 µg mL–1 CB for 20 min); T3 (removal of cumulus cells and pre-incubation in CB (as above)); and T4 (control, no removal of cumulus cells and no pre-incubation in CB). After each treatment, half of the oocytes was washed and immediately cultured for IVM (28 h; denuded oocytes being co-cultured with an equal number of fresh COCs to circumvent the absence of cumulus cells). The other half was exposed to 10% (v/v) ethylene glycol (EG) + 10% (v/v) DMSO for 30 s, followed by exposure to VS (20% EG + 20% DMSO + 0.5 m sucrose) for 20 s at room temperature. Oocytes then were vitrified in <1 µL of VS at the tip of a plastic gutter (2 oocytes/gutter) by direct plunge into liquid nitrogen. After 1 day of storage, vitrified oocytes were warmed in 0.25 m sucrose, extensively washed, and cultured for IVM as described above. All oocytes were then fixed and Hoechst-stained to assess their chromatin status. Oocytes subjected to T1, T2, T3, and T4 without vitrification exhibited no degenerated chromatin (clumped or dispersed) after IVM. After vitrification, the incidence of degenerated chromatin was different (P < 0.05; ANOVA) among treatments, with a lower percentage (P < 0.05) when oocytes were subjected to T3 (15.5 � 3.9%; mean � SD) compared to T1 (26.6 � 3.1%), T2 (48.9 � 3.8%), or T4 (71.1 � 3.8%). The percentage of vitrified oocytes reaching the metaphase II stage (MII) also was different (P < 0.05) among treatments with a higher incidence (P < 0.05) in the T3 group (57.8 � 3.9%) compared to T1 (44.6 � 3.7%), T2 (31.1 � 7.7%), or T4 (15.5 � 3.9%) counterparts. Percentages of non-vitrified oocytes reaching the MII were not different (P > 0.05) among treatments (range, 84.7 to 89.0%) but were higher (P < 0.05) compared to vitrified oocytes. The combination of removal of cumulus cells and pre-incubation in CB prior to vitrification had a cumulative, beneficial influence on cat oocyte survival. Interestingly, removal of cumulus cells imparted a greater benefit on oocyte survival than CB pre-incubation alone.
73 SUPPLEMENTATION OF FROZEN BOAR SEMEN WITH β-MERCAPTOETHANOL INCREASES THE INCIDENCE OF INTACT CELLS AFTER THAWING
Authors: Funahashi H. , Yamaguchi S. , Fujii W. , Murakami T.
Published: January 2008

During the process of freezing and thawing of boar spermatozoa, a large number of the cells appear to be injured by some stresses such as osmotic forces and oxidation, causing reduced viability and penetrability. β-Mercaptoethanol (bME), a strong reducing agent, may ease oxidative stress and rescue sperm cells from those injuries. The aim of this study was to determine the effect of the presence of bME during freezing and thawing of boar spermatozoa on the viability and acrosome status of the sperm cells. Semen samples were collected from 3 boars; only samples with a high motility (more than 80%) were used for this experiment. Each sample was diluted 1:1 with modified Modena solution and kept overnight at 15�C. After centrifugation at 800g for 10 min, the diluent supernatant was removed; spermatozoa were re-suspended at 2 � 109 cells mL–1 in the first diluent (8.8% trehalose solution containing 20% egg yolk and antibiotic) supplemented with 0, 25, or 50 µm bME, and then cooled to 5�C over 2–3 h. At 5�C, semen samples were further diluted 1:1 with the second diluent (same as the first diluent + 5% glycerin + 1.48% Orvus ES Paste (Equex STM; Minitube, Verona, WI, USA)) supplemented with 0, 25, and 50 µm bME, respectively. After packaging the semen into 0.5-mL straws, it was frozen by keeping the straws 4 cm above the surface of liquid nitrogen for 15 min and then storing them in liquid nitrogen until use. After thawing at 37�C for 30 s, semen samples were re-suspended in 10 mL of BTS solution containing 1.15 mm caffeine and 4 mm Ca chloride, and incubated at 37�C under 5% CO2 in air for 90 min. Viability, DNA fragmentation, and acrosome status of spermatozoa were assessed by flow cytometry after staining with SYBR�14/PI (Molecular Probes, Inc., Eugene, OR, USA), acridine orange, and PNA/PI, respectively. Statistical analyses of data from at least 3 replicated trials were carried out by ANOVA and Fisher's protected least-squares difference (PLSD) post-hoc test. Just after thawing, no differences in viability (45.6–51.1%; P = 0.67), DNA fragmentation (0.7–0.9%; P = 0.76), and acrosome status (intact acrosome: 79.2–83.0%; P = 0.26) of the spermatozoa were observed when sperm cells were frozen and thawed in 0, 25, and 50 µm bME. After culture for 90 min, however, the incidence of spermatozoa with an intact acrosome was significantly higher (P < 0.05) when the semen was frozen and thawed in the presence of 50 µm bME (70.9%), compared with 0 (61.7%) and 25 µm bME (61.0%). Chlortetracycline (CTC) analyses were peformed to confirm that the incidence of intact spermatozoa was higher (P < 0.01) in 50 µm bME (67.6%) than that of non-supplementation controls (51.4%). These results demonstrate that supplementation of semen with 50 µm bME during freezing and thawing processes reduces acrosome damage of boar spermatozoa.
76 COMPUTER-ASSISTED SPERM ANALYSIS OF FROZEN SPERM MOTILITY AFTER DIFFERENT TIMES OF EXPOSURE AT 15°C
Authors: Garcia Guerra A. , Etcheverry M. P. , Rodriguez D. , Larraburu G. , Brogliatti G. M.
Published: January 2008

One of the key factors for successful long-term cryopreservation in liquid nitrogen is maintaining the samples at –130°C or lower at all times to avoid cell damage (Barth 1991 Proc. 10th Ann. Conv. Am. Embr. Transf. Assoc., 20–26). Previous data indicated that exposure of the semen straw to ambient temperature for more than 15 s can raise the temperature above –130°C and reduce sperm motility, as determined by subjective evaluation (Berndtson et al. 1976 Proc. 6th NAAB Tech. Conf. Artif. Insem. Reprod., 51–60). The computer-assisted semen analysis (CASA) system provides an opportunity to assess multiple motility characteristics on a semen sample objectively and with high repeatability. An experiment was designed to evaluate the effect of exposing frozen semen in 0.5-mL straws to room temperature for 15, 30, 60, or 120 s on motility characteristics assessed by CASA system. Twenty-eight ejaculates from different bulls (19 Angus, 7 Hereford, 1 Brangus, 1 Shorthorn) were diluted using a chemically semi-defined media (Andromed, Minitüb, Tiefenbach, Germany) and frozen in an automatic freezer (Digicool, IMV, Paillette Crista, France). Five frozen straws per bull were used, one for each time of exposure and one as control (0 s = 0 time). Straws were exposed to room temperature (15°C ± 0.78) for different times and then placed back into liquid nitrogen. Semen thawing was conducted in a water bath at 37°C for 1 min. Motility characteristics were evaluated by the IVOS SpermAnalyzer (Hamilton Thorne Research, Beverly, MA, USA). Two chambers of 20-μm depth and 5 fields per chamber were analyzed (30 frames/0.5 s for each field). Seven motility parameters were evaluated: motile sperm (%), progressive sperm (%), VAP (path velocity, μm s–1), VCL (track speed, μm s–1), ALH (lateral amplitude, μm), BCF (beat frequency, Hz), and LIN (Linearity, %). The Kruskal–Wallis test was used to compare variables among groups, and results are shown in Table 1. There is a significant difference (P < 0.05) in the % of motile and progressive sperm when time of exposure was increased. There was a drastic and significant reduction in the percentage of motile and progressive sperm when exposure to 15°C was longer than 30 s. The live cells had similar motile characteristics: VAP, VCL, ALH, BCF, and LIN. In conclusion, sperm motility would be affected if straws are exposed for more than 30 s. More research should be done to test higher room temperatures, detect viability effects, and determine pregnancy rates after AI.

Table 1. CASA of frozen sperm motility characteristics at different times of exposure at 15°C

This research was supported by Centro Genetico Bovino Eolia S.A.